Cell separation using electric fields

ABSTRACT

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/148,620,filed Sep. 4, 1998, now issued as U.S. Pat. No. 6,043,066 granted Mar.28, 2000, which claims priority from provisional specification60/057,809 filed Sep. 4, 1997, the subject matter of which isincorporated herein by reference.

This invention was made with government support under subcontract no.04027 awarded the National Technology Transfer Center at Wheeling JesuitUniversity supported by the Ballistic Missile Defense Organization,Technology applications Program—NASA Cooperative Agreement no. NCCW-0065. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods and apparatuses for selecting specificcell types from cell suspensions, specifically those employing appliedelectric fields.

BACKGROUND OF THE INVENTION

The ability to isolate specific sub-populations of cells from cellsuspensions is of critical importance to many applications in thebiological sciences as well as to many therapies in clinical medicine.For example, the basis of many medical therapies for treating a varietyof human diseases and for countering the effects of a variety ofphysiological injuries involves the isolation, manipulation, expansion,and/or alteration of specific biological cells. One particularlyimportant example involves the reconstitution of the hematopoieticsystem via bone marrow or progenitor cell transplantation. More specificexamples include: autologous, syngeneic, and allogenic stem celltransplants for immune system reconstitution following the myeloablativeeffects of severe high dose chemotherapy or therapeutic irradiation;severe exposure to certain chemical agents; or severe exposure toenvironmental radiation, for example from nuclear weapons or accidentsinvolving nuclear power generators.

Intensive chemotherapy and/or irradiation for the treatment of a varietyof cancers, including breast cancer, has become a commonly used approachin cancer care centers. Such treatments are associated with severeablation of the bone marrow cells required for function of the blood andimmune systems. Such bone marrow cells are derived from a small numberof progenitor cells known as hematopoietic stem cells in the normal bonemarrow. Therefore, patients receiving such therapies require life-savingtransplants of stem cells in order to survive the effects of thetreatment. Stem cell containing tissue for transplant may be derivedfrom donor marrow (allogeneic transplant) or from the patient's own bonemarrow or peripheral blood after mobilization (autologous transplant).In both instances, there is a need for effective cell separation methodsto enrich the transplant tissue in stem cells and reduce the number ofundesirable and deleterious cells (e.g. mature T cells for allogeneictransplants and residual cancerous cells for autologous transplants).For example, for autologous adjuvant stem cell transplant therapyfollowing myeloablative cancer treatments, it is believed thatreinfusion of residual tumor cells is a major cause of post therapyrelapse. Clearly, removing such cells from transplanted tissue would bebeneficial to the patient.

A number of cell isolation, cell separation, and cell purging strategieshave been employed in the prior art for purifying or removing cells froma suspension. Prior art cell separation methods used to isolate cells orpurge cell suspensions typically fall into one of three broadcategories: physical separation methods typically exploit differences ina physical property between cell types, such as cell size or density(e.g. centrifugation or elutriation); chemical-based methods typicallyemploy an agent that selectively kills or purges one or more undesirablecell types; and affinity-based methods typically exploit antibodies thatbind selectively to marker molecules on a cell membrane surface ofdesired or undesired cell types, which antibodies may subsequentlyenable the cells to be isolated or removed from the suspension. Whilephysical separation methods can be advantageous with regard to theirability to separate cells without causing undo damage to desired cells,current physical separation methods typically have relatively poorspecificity and do not typically yield highly purified or highly purgedcell suspensions. While many chemical and affinity methods have betterselectivity than typical physical methods, they can often be expensiveor time consuming to perform and can cause considerable damage to, oractivation of, desired cells, for example stem cells, and/or can addundesirable agents to the purified or isolated cell suspensions (e.g.toxins, proliferation-inducing agents, and/or antibodies). An additionalpotential problem with antibody-based cell separation techniquestypically employed for purification of stem cells, is that they selectstem cells solely on the basis of cell surface markers (e.g., CD34) andwill not select cells lacking such markers.

In addition to cancer therapy, there are a number of other importantmedical therapies which exist, or are under development, that are basedon cells derived from a variety of different types of stem cells.Examples include pre-exposure prophylaxis or post-exposure therapiesunder development for a variety of biological exposures that may occurnaturally (e.g., viral exposure for example with Ebola, etc.) or beinflicted by mankind (i.e., biological warfare agents). A variety ofgene therapies involving genetically manipulated stem cells, are beingcontemplated or are under development for treating a variety ofblood-related diseases (e.g., AIDS, leukemia, other cancers, etc.). Genetherapy techniques based on genetically manipulated stem and/or germcells may also be useful in cloning organisms, such as animals. However,genetically manipulating stem cells using many current technologies isdifficult, typically employing viruses or gene carriers that can be timeconsuming and expensive, or may be dangerous to perform and may not havehigh yields. Current research findings also suggest that the practicalimplementation of animal organ transplants into human recipients alsomay require procedures involving stem cells from both the donor andrecipient. Many of these promising therapies would requirecryopreservation and storage of donor specimens including human stemcells, for example, as derived from the stem cell-rich umbilical cordblood of newborns, which can provide such donors with a therapeuticbasis for hematopoietic reconstitution or gene therapy should a healthemergency occur later in life. If such storage demands are to berealistically met, the specimens will need to have minimal volume, and,therefore, successful implementation of such technologies may rest onthe development and availability of effective methods for isolatingtrace numbers of stem cells from sources such as umbilical cord bloodand the fetal liver. In order to achieve broad implementation of thetherapies discussed above and others, rapid and cost effective methodsare needed to isolate, with high purity, desired target cells fromsuspensions having a diverse mix of cell types and concentrations.

The use of applied electric fields to physically manipulate cells isknown. Applied electric fields have been employed in the prior art forcell inactivation and sub-lethal cell membrane electroporation. Forexample, U.S. Pat. 5,048,404 to Bushnell discloses a system and methodfor sterilizing liquid foodstuffs by killing microorganisms withexposure to pulsed electric fields.

Sale and Hamilton (“Effects of High Electric Fields on Microorganisms I.Killing of Bacteria and Yeasts,” Biochim et Biophys Acta, 148:781(1967); and “Effects of High Electric Fields on Microorganisms II.Mechanism of Action of the Lethal Effect,” Biochim et Biophys Acta,148:789 (1967)) studied the effect of pulsed electric fields onsuspensions of bacteria or suspensions of yeasts. Specifically, theyinvestigated the effect on the degree of cell kill by the field as afunction of field strength and exposure time. The effect of pulsedelectric fields on the killing of bacteria was also studied by Hülshegeret al. (“Lethal Effects of High-Voltage Pulses on E. Coli K12,” RadialEnviron Biophys, 18:281(1980); and “Killing of Bacteria with ElectricPulses of High Field Strength,” Radial Environ Biophys, 20:53(1981)).Hülsheger et al. studied the effects on bacterial cell death of avariety of experimental parameters and were able to demonstrate a 99.9%reduction in the number of living bacterial cells in suspensions afterexposure to certain pulsed electric field parameters.

The lysis of erythrocytes in erythrocyte suspensions by pulsed electricfields has also been studied both for bovine (Sale and Hamilton,“Effects of High Electric Fields on Microorganisms III. Lysis ofErythrocytes and Protoplasts,” Biochim et Biophys Acta, 163:37 (1967))and human (Kinosita and Tsong, “Voltage-Induced Pore Formation andHemolysis of Human Erythrocytes,” Biochim et Biophys Acta, 471:227(1977); and Kinosita and Tsong, “Hemolysis of Human Erythrocytes by aTransient Electric Field,” Proc Natl Acad Sci. 74:1923(1977))erythrocytes. Knowledge derived from the studies above indicates thatapplied electric fields resulting in cellular transmembrane potentialson the order of 1 Volt can result in colloidal osmotic lysis of theerythrocytes.

Electric fields have also been used to sublethally porate the plasmamembrane of nucleated cells, such as leukocytes and Chinese HamsterOvary (CHO) cells (Sixou and Teissié, “Specific Electropermeabilizationof Leukocytes in a Blood Sample and Application to Large Volumes ofCells,” Biochim et Biophys Acta, 1028:154 (1990)). Sixou and Teissiéinvestigated electropermeabilization conditions to enable reversibleporation of cell membranes, while maintaining long-term cell viability,for the purpose of enabling the reversibly porated cells to uptake drugsand act as immunocompatible drug delivery vehicles within the body.Sixou and Teissié studied the effect of pulsed electric field parameterson the reversible poration of suspensions comprising single cell typesand suspensions comprising mixtures of two cell types (e.g. CHO cellsand erythrocytes, and leukocytes and erythrocytes). The authors showedthat reversible electropermeabilization is a function of the cell sizeand that large cells are reversibly porated at lower electric fieldstrengths than small cells.

While the above mentioned methods and systems for cell separation andcell electropermeabilization represent, in some cases, valuable anduseful techniques for some applications, there remains a need in the artfor simple, fast, and clean methods to selectively isolate or removespecific cell sub-populations from cell suspensions without causing undodamage or activation to the remaining cells and without employingundesirable or toxic agents.

SUMMARY OF THE INVENTION

Accordingly, the present invention can provide relatively simple, fast,and clean methods for cell isolation or purging based on physicaldifferences between different cell types present in a suspension.Furthermore, the invention provides systems and methods that enableselective isolation of viable cells, selective cell inactivation, aswell as stem cell electropermeabilization, using applied electricfields.

In one aspect, the invention provides a method for creating from abiological sample having a given cell population, a suspension of cellsthat contain a selected viable subpopulation of the given cellpopulation. The method is based on a characteristic electroporationthreshold of the cells. The subpopulation of cells selected by themethod is substantially limited to cells that have a characteristicelectroporation threshold that is greater than a predeterminedelectroporation threshold. The selected suspension of cells is producedfrom the biological sample by first subjecting the sample to an electricfield that has a magnitude that is sufficient to porate a substantialfraction of the cells in the sample that have a characteristicelectroporation threshold less than the predetermined electroporationthreshold. The electric field, however, does not porate a substantialfraction of cells that have a characteristic electroporation thresholdgreater than the predetermined electroporation threshold. Essentially,all of the porated cells in the sample that is subjected to the electricfield are also inactivated.

In another aspect, the invention provides a method for creating aselected subpopulation of discreet objects from a sample having a givenpopulation of discreet objects. A discreet object comprises an innerconductive core which is surrounded by a dielectric membrane. The methodis based on a characteristic electroporation threshold of the discreteobjects. The subpopulation of discrete objects selected by the method issubstantially limited to discrete objects that have a characteristicelectroporation threshold that is greater than a predeterminedelectroporation threshold. The selected suspension of discrete objectsis produced from the sample by first subjecting the sample to anelectric field that has a magnitude that is sufficient to causeirreversible dielectric breakdown of the dielectric membrane of asubstantial fraction of the discrete objects in the sample that have acharacteristic electroporation threshold less than the predeterminedelectroporation threshold. The electric field, however, does not causeirreversible dielectric breakdown of the dielectric membrane of asubstantial fraction of cells that have a characteristic electroporationthreshold greater than the predetermined electroporation threshold.

In yet another aspect, the invention provides a method for poratingcells. The method includes supplying a suspension of cells in atreatment volume, where the treatment volume includes at least twoelectrodes that are in fluid contact with the suspension. The methodfurther involves applying a time varying bi-polar electrical potentialacross the electrodes that is sufficient to create an electric fieldthat is sufficient to porate at least one cell in the suspension. Thebi-polar electrical potential is varied so that the average currentacross the sample over the entire treatment time is essentially zero.

In another aspect, the invention provides the method for reversiblyporating stem cells. The method involves supplying in a treatment volumea suspension of cells including a plurality of stem cells, which stemcells have a characteristic size, a characteristic shape, a plasmamembrane, and a nuclear membrane. A pulsed electric field that has apulse duration and magnitude sufficient to porate the plasma membrane ofa cell having a characteristic size and shape essentially identical tothe stem cells, but having an effective membrane thickness substantiallyexceeding the average membrane thickness of the plasma membrane of thestem cells is then applied to the suspension.

In another aspect, the invention involves a system for creating from abiological sample having a given cell population, a suspensioncontaining a selected viable subpopulation of the given cell population.The selected cell population is substantially limited to cells that havea characteristic electroporation threshold greater than a predeterminedelectroporation threshold. The system functions by inactivating asubstantial fraction of the cells in the sample not included in theselected subpopulation. The system includes a generating mechanism thatgenerates an electric field of a magnitude and duration sufficient toirreversibly porate a substantial fraction of the cells not included inthe selected subpopulation, while not irreversibly porating asubstantial fraction of the cells included in the selectedsubpopulation. The system further includes a treatment cell that iselectrically connected to the generating mechanism and is adapted tocontain a cell suspension.

In yet another aspect, the invention provides a system for selectivelyinactivating biological cells based on a difference in a characteristicelectric poration threshold. The system includes a generating mechanismthat generates an electric signal constructed and arranged to createdesired electric field parameters. The system also includes a treatmentcell that is electrically connected to the generating mechanism,includes at least one electrode, and includes a treatment volume adaptedto contain a cell suspension. The electrode is in fluid contact with thecell suspension during operation of the system and is constructed of aporous, biocompatible material, which is sealed in order to reduce therelease of gases from the electrode during operation of the system.

In another aspect, the invention involves a cell suspension comprising aplurality of biological cells suspended in a liquid. The suspensionincludes one population of cells, which have a maximum of characteristicsize not more than a predetermined value, that are substantially viable,and another population of cells, having a maximum characteristic sizegreater than the predetermined value, that are substantially non-viable.The cell suspension is obtained from a precursor suspension ofsubstantially viable cells that contains as subpopulations the two cellpopulations mentioned above. The cell suspension is obtained bysubjecting the precursor cell suspension to an electric field having amagnitude and duration that is sufficient to irreversibly porate asubstantial fraction of the cells in the precursor suspension that havea maximum characteristic size above the predetermined value.

In yet another aspect, the invention involves a cell suspensioncomprising a plurality of biological cells suspended in a liquid whereeach of the biological cells is enclosed by a plasma membrane. The cellsuspension includes a subpopulation of biological cells that possess amaximum characteristic size in excess of a predetermined value.Furthermore, the cells in the subpopulation of cells having a maximumcharacteristic size in excess of the predetermined, value also have amaximum transmembrane electrical potential that exceeds that required tocause irreversible dielectric breakdown of the plasma membrane of thecells.

In another aspect, the invention provides a cell suspension comprising aplurality of non-cultured biological cells, including a plurality ofviable stem cells that have a given characteristic size, suspended in aliquid. The cell suspension further includes a plurality of irreversiblyporated cells, essentially all of which irreversibly porated cells havea characteristic size that is greater than the characteristic size ofthe stem cells.

In another embodiment, the invention provides a cell suspensionincluding a plurality of viable, reversibly electroporated stem cells.

In yet another aspect, the invention involves a suspension comprisingviable, human pluripotent lympho-hematopoietic stem cells, which arecapable of differentiating into members of the lymphoid, erythroid, andmyeloid lineages. The suspension is essentially free of mature andlineage committed cells and is derived from a precursor cell suspensioncomprising substantially viable cells. The suspension is derived fromthe precursor suspension by subjecting the precursor suspension to anelectric field of sufficient duration and magnitude to inactivate asubstantial fraction of the mature and lineage committed cells in theprecursor suspension.

Other advantages, novel features, and objects of the invention will bebecome apparent from the following detailed description of the inventionwhen considered in conjunction with the accompanied drawings, which areschematic and which are not intended to be drawn to scale. In thefigures, each identical or nearly identical component is illustrated invarious figures is represented by a single numeral. For purposes ofclarity, not every component is labeled in every figure.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1a is a schematic illustration showing a cross-section of a cellsuspended in an electric field between two electrodes;

FIG. 1b is a schematic illustration of a portion of the cell membranefrom the cell in FIG. 1 for an applied electric field strength of zero;

FIG. 1c is a schematic illustration of a portion of the cell membranefrom the cell in FIG. 1 for an applied electric field strength less thanthe critical applied electric field strength;

FIG. 1d is a schematic illustration of a portion of the cell membranefrom the cell in FIG. 1 for an applied electric field strengthapproximately equal to the critical applied electric field strength;

FIG. 1e is a schematic illustration of a portion of the cell membranefrom the cell in FIG. 1 for an applied electric field strength exceedingthe critical applied electric field strength;

FIG. 2a is a schematic illustration showing a cross-section of a typicalstem cell;

FIG. 2b is an electrical circuit diagram illustrating how a cell chargesin response to an applied electric field;

FIG. 3 is a flow chart outlining the steps of certain embodiments of theinventive method;

FIG. 4 is a schematic illustration of a batch treatment system accordingto one embodiment of the invention;

FIG. 5 is a schematic illustration showing a cross-sectional view of anelectrode enclosure assembly according to one embodiment of theinvention;

FIG. 6 is a schematic illustration showing a perspective view of adisassembled test cell according to one embodiment of the invention.

FIG. 7 is a schematic illustration of a continuous flow treatment systemaccording to one embodiment of the invention;

FIG. 8 is a schematic block diagram showing the components of a pulsedriver system according to one embodiment of the invention;

FIG. 9 is a graph showing a bipolar electric field pulse according toone embodiment of the invention;

FIG. 10 is a graph showing a unipolar electric field pulse according toone embodiment of the invention;

FIG. 11 is a graph showing surviving cells as a function of PEF electricfield exposure time and magnitude for cells treated according to oneembodiment of the invention;

FIG. 12a is a viability scatter plot derived from flow cytometry datafor a control cell specimen;

FIG. 12b is a viability scatter plot derived from flow cytometry datafor a PEF-treated cell specimen for cells treated according to oneembodiment of the invention;

FIG. 12c is a viability scatter plot derived from flow cytometry datafor a PEF-treated cell specimen for cells treated according to oneembodiment of the invention;

FIG. 12d is a viability scatter plot derived from flow cytometry datafor a PEF-treated cell specimen for cells treated according to oneembodiment of the invention;

FIG. 13a is a scatter plot of data obtained with flow cytometry for acontrol cell suspension;

FIG. 13b is a scatter plot of data obtained with flow cytometry for aPEF-treated cell suspension for cells treated according to oneembodiment of the invention;

FIG. 13c is a scatter plot of data obtained with flow cytometry for aPEF-treated cell suspension for cells treated according to oneembodiment of the invention;

FIG. 13d is a scatter plot of data obtained with flow cytometry for aPEF-treated cell suspension for cells treated according to oneembodiment of the invention;

FIG. 14 is a graph showing surviving cells as a function of PEF electricfield exposure time and magnitude for cells treated according to oneembodiment of the invention;

FIG. 15a is a forward scatter histogram derived from flow cytometry datafor monocytes in a control cell suspension;

FIG. 15b is a forward scatter histogram derived from flow cytometry datafor monocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15c is a forward scatter histogram derived from flow cytometry datafor monocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15d is a forward scatter histogram derived from flow cytometry datafor monocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15e is a forward scatter histogram derived from flow cytometry datafor monocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15f is a forward scatter histogram derived from flow cytometry datafor lymphocytes in a control cell suspension;

FIG. 15g is a forward scatter histogram derived from flow cytometry datafor lymphocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15h is a forward scatter histogram derived from flow cytometry datafor lymphocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15i is a forward scatter histogram derived from flow cytometry datafor lymphocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 15j is a forward scatter histogram derived from flow cytometry datafor lymphocytes in a PEF-treated cell suspension for cells treatedaccording to one embodiment of the invention;

FIG. 16 is a graph showing lymphocyte enrichment as a function ofelectric field exposure time and magnitude for cells treated accordingto one embodiment of the invention;

FIG. 17 is a bar chart showing surviving fraction of cells and stem cellenrichment as a function of electric field strength for cells treatedaccording to one embodiment of the invention;

FIG. 18a is a light scatter plot derived from flow cytometry data forinput CMK cells to be treated according to one embodiment of theinvention;

FIG. 18b is a viability stain histogram derived from flow cytometry datafor input CMK cells to be treated according to one embodiment of theinvention;

FIG. 18c is a light scatter plot derived from flow cytometry data forinput PBMC cells to be treated according to one embodiment of theinvention;

FIG. 18d is a viability stain histogram derived from flow cytometry datafor input PBMC cells to be treated according to one embodiment of theinvention;

FIG. 18e is a light scatter plot derived from flow cytometry data for aninput CMK/PBMC cell mixture to be treated according to one embodiment ofthe invention;

FIG. 18f is a viability stain histogram derived from flow cytometry datafor an input CMK/PBMC cell mixture to be treated according to oneembodiment of the invention;

FIG. 19a is a light scatter plot derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 19b is a bivariate plot derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 19c is a viability histogram derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 19d is a light scatter plot derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 19e is a bivariate plot derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 19f is a viability histogram derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 20a is a light scatter plot derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 20b is a bivariate plot derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 20c is a viability histogram derived from flow cytometry data for acontrol cell specimen containing a CMK/PBMC cell mixture;

FIG. 20d is a light scatter plot derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 20e is a bivariate plot derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 20f is a viability histogram derived from flow cytometry data for aPEF-treated cell specimen containing a CMK/PBMC cell mixture for cellstreated according to one embodiment of the invention;

FIG. 21 is a graph showing surviving cells as a function of PEF electricfield magnitude for three types of cells for cells treated according toone embodiment of the invention;

FIG. 22 is a graph showing surviving cells as a function of PEF electricfield magnitude and pulse duration for cells treated according to oneembodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods and systems for selectivelyinactivating biological cells, or any discrete objects having an innerconducting core surrounded by a dielectric layer, for example a lipidbilayer membrane. Specifically the invention provides methods andapparatus for selectively inactivating such cells or discrete objects bysubjecting a suspension containing such cells or discrete objects to anapplied electric field of sufficient duration and magnitude to causedielectric breakdown or electroporation of the dielectric layer. Themethods provided by the present invention can be used advantageously toselectively inactivate subpopulations of cells or discrete objects froma precursor population that contains a mixture of different cells ordiscrete objects, or mixtures of cells and non-cell discrete objects, onthe basis of a characteristic electroporation threshold, thus providinga means for selectively purging or isolating cells or discrete objectsfrom larger populations. One embodiment of the inventive method involvessubjecting a sample having a given population of cells or discreteobjects to electric field conditions sufficient to porate a substantialfraction of cells or discrete objects that have a characteristicelectroporation threshold below a selected predetermined value, whilenot simultaneously porating a substantial fraction of the cells ordiscrete objects having a characteristic electroporation above thepredetermined value, and subsequently, or simultaneously inactivatingthe porated cells or discrete objects.

The term “biological cell” or “cell” as used herein has its commonlyunderstood meaning and includes viable, potentially viable, orpreviously viable cells derived from a biological sample. Such cellsinclude prokaryotic cells such as bacteria, and algae, and eukaryoticcells, such as yeasts, fungus, plant cells, and animal cells. Such cellstypically have an inner, electrically conducting core comprised ofcytoplasm, surrounded and enclosed by at least one dielectric membrane,for example the cytoplasmic or, equivalently, plasma membrane.Eukaryotic cells, in addition, typically also possess a dielectricnuclear membrane surrounding a conductive nucleus within the interior ofthe cell.

The term “discrete objects having an inner conducting core surrounded bya dielectric layer” or simply “discrete object” as used herein refers toany object comprising a substance exhibiting a relatively low electricalresistivity surrounded and enclosed by a dielectric layer or dielectricmembrane having a much higher electrical resistivity. Such discreteobjects include biological cells as previously described, but alsoinclude objects such as certain viruses, sub-cellular organelles,liposomes, micelles, and others. Throughout the remainder of thisdetailed description, many of the methods and apparatus of the inventionare described in relation to biological samples comprising biologicalcells. It should be understood that the invention is not so limited andthat the invention may similarly be applied to discrete objects, asdefined herein, other than cells. Also, whenever the term “discreteobject” is used herein, it should be understood that the term includes,as a subset, biological cells. The term “dielectric layer,” or“dielectric membrane,” or “membrane” as used herein refers to acontinuous layer or coating having a finite thickness and having anelectrical resistivity (Ω·cm) exceeding that of a conducting core whichthe membrane encloses. Typically, the electrical resistivity of thedielectric layer will exceed that of the inner conducting core by atleast a factor of 10, and more typically, for example as is the casewith most biological cells, by at least a factor of 10⁴-10⁹. In thecontext of biological cells, the dielectric layer is defined by at leastone lipid bilayer membrane, together with any associated structures orsubstances associated therewith which affect the effective membranethickness or resistivity of the dielectric layer. “Effective,” as usedherein in the context of membrane thickness or resistivity, refers to athickness or resistivity of an equivalent membrane not possessing anyassociated structures or substances affecting its dielectric propertiesassociated therewith that possesses the same dielectric properties asthe actual membrane having such associated structures or substances.

The term “inactivating” as used herein refers to destruction of at leastone property of a discrete object. In the context of biological cells,inactivating is equivalent to rendering unviable, or killing the cell.As applied to non-cell discrete objects, inactivate can refer tophysical destruction of the object, or simply a destruction of thepermi-selective diffusional barrier properties of the dielectric layerwith respect to at least one molecular, ionic, or atomic species. Incertain embodiments involving cells, inactivation may involve not onlyrendering the cells non-viable, but also irreparably lysing andphysically disrupting and destroying the physical structure of the cell.

The invention provides, in some embodiments, relatively fast andeffective methods for purifying certain desired cells in a viable statefrom a suspension or eliminating certain undesired cells from a cellpopulation. As mentioned, the methods involve exposing a suspensioncontaining a population of cells to an applied electric field, whichfield has a magnitude and is applied for a duration selected to porate,and in some embodiments inactivate, a substantial fraction of certainsubpopulations of cells based on their characteristic electroporationthreshold. The term “suspension of cells” as used herein refers to amixture of cells suspended in a carrier liquid. The carrier liquid maybe naturally part of the biological sample from which the cells derive,for example blood is a suspension of blood cells suspended in plasma,or, for cells which are not normally present in a suspension, thecarrier liquid can be any suitable diluent or medium. Preferred carrierfluids are non-toxic and physiologically compatible with the cells theysuspend, at least for a time period equal to that of the electric fieldapplication procedure. Preferred carrier fluids are also electricallyconductive. The electrical conductivity can be any value greater thanzero, but preferably will range from about 10%-200% that of theconductive core of the cell. In certain embodiments, the conductivity orresistivity of the carrier fluid will be essentially equal to that ofthe conductive core of the cell. In other embodiments, in order toreduce the power consumption of the electric field generating apparatusand/or reduce the degree of heating of the cell suspension, as willdiscussed in greater detail herein, it can be preferable to utilize acarrier fluid having a resistivity that is greater than that of theconductive core of the cell. In order to provide a more uniformelectrical field throughout a cell suspension being treated according tothe invention, the cell suspension should be essentially free of gasbubbles. The cells in the suspension should also be individuallysuspended and as free from clumping and aggregation as possible duringapplication of the electric field in order to provide the maximumresolution and selectivity attainable for a given set of electric fieldexposure conditions. The concentration of cells in a cell suspension, aswill be discussed in more detail, can also affect the selection andperformance of the applied electric field. Typically, for mammaliancells, the range of total cell concentrations in treated samples canrange from about 10²-10¹⁰ cells/ml, with preferred suspensions havingfrom about 10⁴-10⁸ cells/ml.

The term “substantial fraction” or “substantially depleted” as usedherein, in the context of discrete objects having a characteristicelectroporation threshold less than a predetermined value, refers to atleast 25% of such cells being porated by the applied electric field andinactivated, preferably at least 50%, more preferably at least 90%, insome embodiments preferably at least 99%, in some embodiments preferablyat least 99.99999%, and in some embodiments preferably essentially allof the discrete objects. The term “electroporation threshold” or“critical electroporation threshold” as used herein refers to thesusceptibility of a particular cell or subpopulation to membraneporation by an applied electric field. As will be discussed in greaterdetail herein, the characteristic electroporation threshold of a cell orsubpopulation of cells is a function of the physical and chemicalproperties of the cell that influence its interaction with an appliedelectric field. Differences between one or more of these propertiesbetween different cells or subpopulations may be advantageouslyexploited to provide a basis for selective isolation and inactivationaccording to the invention. Such properties include characteristic cellsize, effective dielectric membrane thickness, cell shape, cell and/ormembrane morphology, cell membrane capacitance, cytoplasmic electricalresistivity, etc. The term “predetermined electroporation threshold” asused herein refers to a chosen value of electroporation threshold belowwhich a significant fraction of cells having such electroporationthresholds will be porated by the applied electric field, and abovewhich a significant fraction of cells having such electroporationthresholds will not be porated. The predetermined electroporationthreshold, as will be discussed in greater detail herein, can be afunction of the parameters of the applied electric field (e.g. fieldstrength, field duration, etc.) and the electrical properties of thecell suspension (e.g. fluid carrier resistivity, total suspensioncapacitance, etc). An important feature of the invention is theselection of such electric field parameters and suspension properties inorder to provide conditions that selectively inactivate a significantfraction of one or more subpopulations of cells in a sample based on oneor more differences in their properties that affect their characteristicelectroporation threshold.

The methods and apparatus provided by the present invention can beapplied to a wide variety of discrete object separation applications,and to a wide variety of biological and non biological samples. Oneimportant embodiment of the invention provides a method of isolating orinactivating discrete objects based on a characteristic size.“Characteristic size” or “size” as used herein with respect todimensions of cells or discrete objects, refers a linear dimension of acell or discrete object as measured in the direction of an appliedelectric field to which the cells or objects are subjected and from anexternal surface of the outermost dielectric membrane on one side of thegeometric center of the cell or object, through the geometric center ofthe cell or object, to an external surface of the outermost dielectricmembrane on the other side of the geometric center of the cell orobject. For example, for a spherical cell or object, the characteristicsize would be the external diameter of the cell or object. Specifically,the method involves porating and inactivating a substantial fraction ofdiscrete objects above a certain predetermined threshold size, which isa function of the electric field and suspension properties as discussed,while leaving in an uninactivated state a significant fraction ofdiscrete objects below the threshold size. The method can be used, forexample, to inactivate cells present in viral preparations used intreatment, diagnosis, or research of viral diseases such as AIDS. It isoften desirable to obtain suspensions of pure virus that are essentiallyuncontaminated with viable cells infected with such virus. Becausevirus-carrying cells will typically be substantially larger thanviruses, the inventive method can be use to selectively inactivate thecells without causing undue damage to the virus. Another applicationinvolves isolation of certain sub-cellular organelles from cells. Inthis case, the cells can initially be porated using the inventive methodin order to liberate the intracellular contents of the cells into thesuspension. Subsequently, an electric field having different parameterscan be applied to the suspension to selectively isolate one or moresubpopulations of organelles on the basis of a difference in acharacteristic electroporation threshold, for example due to adifference in characteristic size. An example of the inventive method asapplied to a non-biological sample is the selective disruption ofliposomes on the basis of size. Liposomes are commonly used as vehiclesfor drug delivery or for transfection of genetic material into cells.The performance of the liposome for its intended task and alsopotentially its pharmacokinetics within the body can be a function ofthe size of the liposome. Thus, the current invention can provide arelatively fast and easy means for performing a size selection ofmanufactured liposomes.

Two important applications where the present invention has particularutility involve the purging of cancer cells from cell suspensions andthe isolation and enrichment of stem cells or germ cells from cellsuspensions. A “germ cell” as used herein, refers to haploid cells, orgametes, such as sperm or egg cells. These applications are importantcomponents in many clinical treatment therapies involving, for example,cancer treatment, organ transplant, and gene therapy. The present methodexploits a difference in a critical electroporation threshold betweenthe above mentioned cell types and the other cell types present in thecell suspension to effect a selection of desired cells and/or a purgingof undesired cells. In particular, in particular embodiments,differences in characteristic cell size (e.g. average cell diameter)provide, at least in part, for the above mentioned difference incritical electroporation threshold. Cancer cells, for example, are oftensignificantly larger than the desired population of cells, for examplestem cells, in a sample and will generally have an electroporationthreshold below that of the desired cells. Thus, by subjecting thesuspension to a selected electric field having predeterminedcharacteristics, a substantial fraction of the cancer cells can beinactivated without inactivating the desirable cells. Preferably, when asuspension initially containing such cancer cells is subjected to theinventive cell selection method, the concentration of viable cancercells remaining is decreased by at least a factor of 10 (1 logreduction) and most preferably by at least a factor of 100,000 (5 logreduction). Similarly, because for many biological samples stem cellsare the smallest cells present, the inventive method can be used toenrich such a sample in viable stem cells by selectively inactivatingnon-stem cells in the sample. Preferably, the concentration of viablestem cells, with respect to the total number of viable cells present inthe sample, is increased in the sample through application of theinventive method by at least a factor of two, more preferably by atleast a factor of five, and in certain preferred embodiments, by afactor of 10⁶ or more, while, correspondingly, the concentration ofviable non-stem cells in the sample is substantially depleted. Onefeature of the inventive method when applied to the purification andenrichment of stem cells, is that unlike typical antibody-based stemcell isolation methods, isolation of stem cells using applied electricfields does not rely on the presence of surface markers on the stemcells (e.g. CD34). The most primitive stem cells may not possess thecell surface markers targeted by typical antibody-based methods andthus, such cells will not be recovered by those methods. Conversely,stem cells isolated by the present methods are selected on the basis oftheir critical electroporation threshold. Therefore, the stem cellsuspensions provided according to the inventive methods, will includestem cells that do not possess the surface markers typically used byantibody-based methods to select stem cells if such stem cells wereinitially present in the sample before application of the inventivemethods. Specifically, one embodiment of the present invention canprovide a suspension enriched in stem cells, which is essentially freeof mature and lineage committed cells, and which includes as asubpopulation, stem cells not expressing CD34 on their surface.

The stem cell and/or cancer cell containing suspensions can be derivedfrom a variety of sources including, but not limited to, bone marrow,mobilized or unmobilized peripheral blood, umbilical cord blood, fetalliver tissue, other organ tissue, skin, nerve tissue, etc. A variety ofstem cells may advantageously be isolated and enriched according to theinvention including, but not limited to, hematopoietic stem cells,embryonic stem cells, mesenchymal stem cells, epithelial stem cells, gutstem cells, skin stem cells, neural stem cells, liver progenitor cells,and endocrine progenitor cells. One embodiment of the invention involvesthe isolation of lympho-hematopoietic stem cells, which are capable ofdifferentiating into members of the lymphoid, erythroid, and myeloidlineages, from cell suspensions including mature and lineage committedcells to provide a suspension of lympho-hematopoietic stem cells that isessentially free of mature and lineage committed cells. The enrichedstem cell suspensions according to the present method will also beadvantageously enriched in pluripotent stem cells, which have theability to differentiate into the full complement of mature cellsderived from a particular type of stem cell. Also, in some embodiments,the enriched stem cell suspensions produced according to the inventionwill contain, in addition to pluripotent stem cells, stem cells whichare committed colony forming cells. For example, for samples includinghematopoietic stem cells, the enriched suspensions can advantageouslyinclude viable colony forming cells for granulocytes and macrophages(CFC-GM), colony forming cells for erythrocytes (BFU-E), colony formingcells for eosinophils (CFC-Eo), multipotent colony forming cells(CFC-GEMM), and immature lymphoid precursor cells.

Thus, it is apparent that the present invention provides a novel methodfor cancer purging and stem cell isolation useful for a variety ofmedical therapies, an important one of which is stem celltransplantation for hematopoietic reconstitution after myleoablativetherapies. A particularly attractive application for the teachings ofthis invention is the isolation of hematopoietic stem cells from bonemarrow, mobilized peripheral blood, umbilical cord blood or fetal livertissue, which is a crucial first step in an overall protocol fordelivering genetically manipulated, stem-cell-based, pathogencountermeasures that have the potential to provide pre-exposureprophylaxes or post-exposure therapies, and immune systemreconstitution. Isolation of stem cells to high purity prior to theirgenetic manipulation is essential for eliminating the interference andcomplications that occur should other leukocytes be present/viableduring gene transfection and expansion. Cryostorage of large numbers ofstem cell specimens will be required for large scale implementation ofsuch stem cell-based-countermeasures. Efficient, practical cryostorageof large numbers of specimens demands small specimen volumes. Since therelative concentrations of stem cells in bone marrow mononuclear cell(BMMC) and mobilized peripheral blood mononuclear cells (MPBMC)specimens are approximately 1:10⁵, stem cell isolation and enrichmentwill be important for achieving small specimen volumes advantageous forefficient cryostorage. To eliminate interference from non-stem cellsduring expansion and gene transfection, and to reduce volumerequirements for cryostorage, isolation strategies should be capable ofenriching stem cell concentrations by up to 10⁶ for BMMC and MPBMCspecimens.

Many presently available methods typically in use for stem cellisolation or cancer cell purging depend on antibody binding to cellsurface structures or toxin-based cell inactivation strategies. Thesestrategies can be sub-optimal for stem cell-enrichment because theyprovide in some cases relatively low degrees of enrichment and can adddetrimental substances to the suspension, such as exogenous antibodiesor toxins, that damage or can activate the isolated stem cells or bedetrimental to a patient upon reinfusion of such cells. Other currentlyavailable stem cell isolation strategies involve a culture-basedprotocol requiring a long processing time, for example up to one week.In addition, many currently available cell isolation methods do noteasily scale, and, therefore, are not optimal for handling the largethroughput required for a widespread implementation of manystem-cell-based therapies. Thus, in the prior art, there exists a needfor stem cell isolation strategies for effective implementation of thestem-cell-based countermeasures, which the present invention, in someembodiments, potentially can fill.

The methods and apparatus provided according to the present inventioncan provide significant improvements and advantages over many prior artmethods for performing cell separations and isolations. The presentmethod is based on intrinsic differences between cell types, for examplecharacteristic size and/or characteristics that effect the membranebreakdown voltage, such as the dielectric strength of the membrane orthe effective membrane thickness, and does not require, in manyembodiments, the addition or use of exogenous agents, such as antibodiesor toxins, which can adversely affect the viability or state ofactivation of the isolated cell fraction. “Dielectric membrane breakdownvoltage” or “membrane breakdown voltage” refers the voltage across thedielectric membrane layer of a cell or discrete object at the onset ofporation of the membrane. In addition, the present method issubstantially faster than many prior art cell separation techniques.Cell inactivation and isolation using the inventive method can beperformed in times ranging from milliseconds to minutes. Finally, withappropriate selection of operating parameters, as discussed herein, androutine optimization of the selected parameters and method, theinventive method can potentially provide high degrees of enrichment orpurging of selected cell subpopulations.

As discussed in more detail later herein, cell selection with electricfields according to the invention can be used as a stand alone method ormay be combined with one or more other cell separation methods, suchother methods being a pre-treatment or post-treatment step. Also themethod according to the invention can be applied to a cell suspension sothat the applied electric field both porates and inactivates one or moresubpopulations of cells in a single step, or, alternatively, the appliedfield may porate some or all of the cells to be inactivated withoutinactivating all of such cells, with the inactivation step performed ina subsequent step. In the former case, the applied electric field issufficient, under the conditions of its application, to causeirreversible breakdown or irreversible poration of the dielectricmembrane of the cell. “Irreversible breakdown” or “irreversibleporation” refers to poration that is sufficient to cause death,inactivation, and/or physical disruption of a discrete object without aneed for a secondary inactivating step. Inactivation and cell death dueto poration are believed to be caused by a loss of the permi-selectivenature of the membrane leading to cell death and/or membrane disruption,or a direct physical disruption of the membrane caused by extensiveporation. In the case of a loss of the permi-selective nature of themembrane, the inactivation or cell death is ultimately caused bydiffusion of previously excluded molecular species, especially smallionic species such as Na⁺, K⁺, and Ca⁺⁺, across the membrane followed byan uptake of water across the membrane into the cell in an attempt toachieve osmotic equilibrium with the suspending fluid medium, which canlead to colloidal osmotic lysis and irreparable (fatal) cell lysis, orto a lethal disruption of cellular metabolism. For embodiments involvinga method where the applied field porates some or all of the cells to beinactivated without inactivating all of such cells, where theinactivation step is performed in a subsequent step, the porationinduced by the applied field is typically less extensive and notirreversible, at least for a certain portion of the porated cells. Givensufficient time, the reversibly porated cells in such samples could sealtheir pores and retain long-term viability if left in the same fluidcarrier or suspending media in which they were subjected to the electricfield. The reversibly porated cells may, however, be effectivelyinactivated by resuspending them in a different post-poration media,adjusting the temperature of the poration media, and/or adding asupplemental agent to the poration media which accelerates cell death,colloidal osmotic lysis, or prevents the resealing of membrane pores.More specific techniques and conditions are discussed later herein.

The electric field is preferably applied to the cell suspension within aspatially defined treatment cell. The treatment cell can be designed asa static non-flow volumetric container in which the cell suspension tobe treated is placed, or more preferably, the treatment cell willinclude an inlet and an outlet constructed and arranged to enable a cellsuspension to continuously flow through the treatment volume. Systemsincluding flow-through treatment cells may be arranged so that the cellsuspension passes through the treatment cell only once (one pass) or aplurality of times (recirculating). In addition, either the flow orstatic systems may include multiple treatment cells. For flow systems,multiple treatment cells can be arranged in a series or parallelconfiguration.

The treatment cell will include at least one electrode in electricalcommunication with the cell suspension to be treated. Preferably thetreatment cell will include two electrodes placed on either side of andin electrical communication with the cell suspension during operation towhich an electric potential is applied to produce an electric fieldwithin the treatment volume. In preferred embodiments, the treatmentcell and electrodes are constructed and arranged to impose an electricfield that is substantially spatially uniform within the treatmentvolume so that all cells in the suspension are exposed to similarelectric field conditions. In some embodiments, the electric fieldapplied to the cell suspension is created by an electric signal appliedto the electrodes; however, it is also contemplated that the electricfield can be induced in the sample cell via induction by a magneticfield.

In order to reduce the tendency for the electrical potential applied tothe electrodes to discharge by arcing, and in order to reduce the degreeof electrical heating that occurs in the cell suspension, in certainpreferred embodiments, the applied electric field is pulsed for shortdurations, such durations, except as otherwise described herein, beingshorter than the residence time of the treated cell suspension in thetreatment volume during the step of subjecting the suspension to theapplied electric field. Such electric fields are hereinafter referred toas “pulsed electric fields” or PEFs. The shape of the electric fieldpulse is preferably substantially rectangular in shape, thus providingvery short voltage rise and fall times and a substantially constantmagnitude over the entire pulse length. Such rectangular pulse shapesyield the best performance and poration threshold resolution obtainablewith the inventive method. While rectangular pulses are preferred, anypulse shape known in the art may be employed in performing the methodsof the invention, especially when high resolution is not required, as,for example, when inactivating a cell type that is substantially largerthan the desired cell type.

As described previously, the electric field parameters required toeffect a desired cell inactivation or isolation depend upon the natureof the cells, the suspension and suspending fluid, and thecharacteristics of the electric field application apparatus. The exactparameters for any given sample that will yield desired results must befound in practice via routine experimentation. What follows herein is atheoretical development and description of the inventive method,apparatus for performing the method, and important parameters affectingthe performance and selectivity of the method to provide guidance tothose of skill in the all in selecting parameters to develop successfulcell or discrete object isolation and inactivation strategies.

The Fundamental Basis of Electric Field Cell Isolation

The mechanism by which electric fields, and particularly pulsed electricfields (PEFs), isolate cells can be best understood by examining theresponse of a single discrete object, as exemplified by a biologicalcell, to an externally applied electric field. A schematic illustrationof such a system 50 is shown in FIGS. 1a-e. The externally appliedelectric field 57 can be established by applying a constant voltage orvoltage pulse across a pair of electrodes 55 and 56 that are inelectrical communication with, and preferably in physical contact with,a cellular suspension containing a plurality of cells, one of which 51is shown in FIG. 1a. Alternatively, the electric field 57 can be appliedinductively by creating a time-varying magnetic field throughout thecellular suspension. To preserve the viability of the desired targetcells, the carrying fluids in which the biological cells are suspendedare typically buffered saline solutions having, in some embodiments, astandard physiological osmolality (e.g. 275-300 mOs/kg-water for mostmammalian cells), and a pH in the physiological range (e.g. about7.0-7.6 for most mammalian cells). The ionic strength of the solutionsin certain embodiments is essentially the same as the ionic strength ofthe intracellular fluid 53 (e.g. about 0.15 M NaCl equivalent for mostmammalian cells). As such, these are conducting solutions. Electricfield effects on cells can be estimated from the potential theorydeveloped by Coulson (Coulson CA: Electricity, Oliver and Boyd, London,Chapter 9, 1951) incorporated herein by reference. This theory impliesthat induced transmembrane potentials depend on cell size and shape.Formally, the external electric field induces a potential across thecell 51, V_(cell), given by $\begin{matrix}{{V_{cell} - {{flE}\quad {where}\quad f}} = {l/\left( {l - {\frac{1}{3}d}} \right)}} & (1)\end{matrix}$

and where E is the field strength of the imposed electric field; d isthe cell diameter 54, l is the projected length of the cell in theelectric field direction 57; and f is a form factor, which is equal to1.5 for a spherical cell (where l is equal to d) and is approximatelyunity for large aspect ratio cylindrically shaped cells (where l>>d). Inthe development to follow, the cells of interest will be assumed to bespherical so that d will be used for l in the following equations and fwill be set equal to 1.5. For a more detailed discussion on the effectsof non-spherical cell shape and angular orientation with the appliedelectric field, the reader is referred to Kinosita and Tsong (Kinosita Kand Tsong TY, Voltage-induced pore formation and hemolysis of humanerythrocytes, Biochim et Biophys Acta. 471:227-242, 1977).

Biological cells have an outer, semipermeable plasma membrane 52 thatallows the cell to control its internal environment by its selectivepermeability. The proper function of this membrane is crucial to theviability of the cell. If the function of this membrane is altered ordestroyed, cell death often follows. Plasma membranes are typicallylipid bilayers which behave electrically as dielectrics, i.e., theybehave as electrical insulators. For eukaryotic cells, as shown in FIG.1a, the cell nucleus 68 and accompanying nuclear membrane 69 residewithin the outer membrane 52, with cytoplasm 53 filling the gap betweenthe nuclear and outer membranes. For prokaryotic cells, there is nonucleus or nuclear membrane, so the cytoplasm, which supports the cell'sgenetic information (one or more DNA molecules in the form of nucleoids)fills the entire intracellular volume. Cytoplasm, which referscollectively to the substance filling the gap 53 between the outer andnuclear membrane for eukaryotic cells, or the entire intracellularvolume for prokaryotic cells, is mainly composed of cytosol, which is asemifluid concentrate having an electrical resistivity that is similarto that of aqueous solutions having a standard physiological ionicstrength. As such, the cytosol is electrically conductive, whichdictates that the intracellular volume of both eukaryotic andprokaryotic cells is electrically conductive. Thus, biological cells canbe viewed as a conducting intracellular region surrounded by adielectric (insulating) membrane 52. With this conceptual view ofbiological cells, application of an external electric field 57 causescharge separation to occur inside the biological cell 51 resulting in anearly constant intracellular potential that has a value correspondingto the boundary average of the potential established on the outersurface of the cell's dielectric membrane 52. If the poles of the cell51, of which there are two, are defined as the two points formed on thesurface of the cell 51 by the intersection of a ray parallel to theelectric field direction passing through the center of the cell, thenapplication of an external electric field causes one half of thepole-to-pole potential drop outside of the biological cell 51 to developacross the membrane 52 at each pole of the cell. That is, the externallyapplied electric field 57 produces a maximum transmembrane potential,V_(m), at each pole of the cell 51 that scales as

V _(m) =V _(cell)/2  (2)

or equivalently

V _(m)=3dE/4  (3)

for a spherical cell.

Since, in response to an externally applied electric field 57, thepotential drop, V_(cell), that develops over a cell's diameter 54 orprojected length is transferred approximately equally across the twopoles of the cell's membrane 52, the maximum electric field therebyimparted to a cell's membrane 52 is

E _(m) =V _(cell)/2t _(m),  (4)

or, for spherical cells

E _(m)=3Ed/4t _(m)  (5)

or equivalently

E _(m) =V _(m) /t _(m)  (6)

where E_(m) is the electric field imparted to the membrane 52 for anexternally applied electric field 57 of strength E; d is the diameter 54of the biological cell 51; and t_(m) is the thickness of the membrane.Thus it is apparent from equation 5, that the imposed electric fieldwithin the cell membrane is directly proportional to cell size andapplied electric field strength and inversely proportional to thethickness of the cell membrane. Since the size of many typicalbiological cells falls within a range of 1<d<50 μm and a typicalthickness of cell membrane 52 lipid bilayer is approximately 5 nm, theelectric field strength imparted to the membrane 52 can be two to threeorders of magnitude greater than the strength of the externally appliedelectric field 57. More specifically, for a typical lipid bilayermembrane thickness of about 5 nm, a transmembrane potential, V_(m) ofapproximately one Volt will impart a 2 MV/cm electric field, E_(m), tothe lipid bilayer membrane 52. So for a 10 μm diameter spherical cell,which, for example, is about the mean size of peripheral blood cells, a2 kV/cm externally applied electric field E would generate the 2 MV/cmelectric field, E_(m), in the lipid bilayer membrane. Since thedielectric strength of many polymers, in response to electric fields, isin the range 0.1-0.5 MV/cm, it is reasonable to expect that a 2 MV/cmelectric field imparted to the membrane 52 of a 10 μm diameter cell byan externally applied 2 kV/cm electric field 57 would produce membranepores by dielectric breakdown. Thus, the electric field magnificationprovided by the electrical behavior of biological cells can lead to thedielectric breakdown of a cell's membrane 52 when the externally appliedelectric field has sufficient strength, thereby forming irreversiblepores in the membrane which lead to cell death. From equations 1 and 5,it is apparent that the susceptibility of a given cell to poration by anapplied electric field 57 is proportional to the magnitude of theapplied electric field, E, that is required to produce a given electricfield, E_(m), in the lipid bilayer membrane (which is directly relatedto membrane poration) and is related to the size of the cells, thethickness of the dielectric membrane, the dielectric strength of themembrane (V/m), the shape of the cells and the orientation ofnon-spherical cells in the applied electric field. Thus, a difference inone or more of these properties between different cell types can lead toa difference in their characteristic electroporation threshold and canpotentially be exploited to effect a selective cell isolation orinactivation using an applied electric field, as described in greaterdetail to follow. These relations are rigorously valid when the cell isimmersed in a conducting fluid, but such fluid may not be required insome embodiments for the inventive method to be functional.

For lipid bilayer membranes, which are typical of many mammalian cellsand bacterial cells, the onset of membrane dielectric breakdown inresponse to an externally applied electric field has been relativelyconsistently observed in the prior art when the transmembrane potential,V_(m), reaches a particular critical value, namely V_(m)=V_(mc), whereV_(mc) is the critical transmembrane potential for dielectric breakdown,or, equivalently, the dielectric membrane breakdown voltage. Table 1summarizes data from cell poration experiments assembled by Castro, etal. (Castro A J, et al: Microbial Inactivation of Foods by PulsedElectric Fields. Washington State University, Department of Food Scienceand Human Nutrition, Pullman, Wash., 99164-6376, 1993) hereinafter“castro”)showing the critical dimensions of various viable cells andtheir critical membrane potentials for cell membrane poration. Thecritical dielectric membrane breakdown voltage V_(mc) for these cellswere obtained by using Eq. 3 together with results from cellinactivation experiments which measured the critical threshold appliedelectric field, E_(c).

TABLE 1 Cell size and induced membrane potential for severalmicroorganisms. d l v V_(mc) Microorganism (μm) (μm) (μm³) f (V) E. coli(4 hr) 1.15 6.9 7.2 1.06 0.26 E. coli (30 hr) 0.88 2.2 1.4 1.15 1.05 K.pneumoniae 0.83 3.2 1.7 1.09 1.26 P. aeruginosa 0.73 3.9 1.6 1.07 1.26S. aureus 1.08 n/a 0.6 1.50 1.00 L. monocytogenes I 0.76 1.7 0.8 1.170.99 C. albicans 4.18 n/a 38.0 1.50 2.63

In the table, v, is the volume of the cells. With the importantexception of young cells (e.g., E. coli, 4 hr culture, in thelogarithmic growth phase), the critical membrane potentials V_(mc) ofcells in their stationary phase is approximately 1 Volt. A variety ofparameters can effect dielectric membrane breakdown voltage, V_(mc).Such parameters include, but are not limited to, the membrane thickness,the dielectric strength of the membrane, the dimensional and chemicaluniformity of the membrane, etc. Since, for a given dielectric material,the threshold transmembrane electric field strength, E_(mc), fordielectric breakdown is often similar, and since most of the cells inTable 1 have similar dielectric membranes (lipid bilayers together withany associated protein and/or carbohydrate components), Eq. 6 wouldsuggest that the electrical properties, specifically the membranethickness, of the membranes having similar V_(mc) are similar. Inaddition to rapidly growing cells, another important exception to thegeneral rule of 1 Volt being a critical transmembrane potential for cellporation and inactivation are spores in their quiescent state which havebeen shown to be much more insensitive to electric fields and appear tobe sensitive only during germination and outgrowth when the cortexdisappears and the spore coat layers dissolve as the cell swells(Hüsheger H., hotel J., and Niemann E-G. Electric Field Effects onBacteria and Yeast Cells, Radiat. Environ. Biophys. 22:149-162, 1983.;(hereinafter “Hulsheger 1983”) and Grahl T., Sitzman W., Markl II.Killing of Microorganisms in Fluid Media by High-Voltage Pulses.Presented at 10th Dechema Annual Meeting of Biotechnologists. Karlsruhe,Germany, Jun. 1-3, 1992.). This behavior may be due to the quiescentspores having a much thicker effective dielectric membrane thickness dueto the presence of the coat layers. Thus for a given criticaltransmembrane electric field strength, E_(mc), which is a function of,for example the resistivity and material properties of the dielectriclayer, a larger effective membrane thickness would, according to Eq. 6,yield a larger V_(mc), and would thus require a larger critical appliedelectric field, E_(c), for cell inactivation (see Eq. 3).

As shown in FIGS. 1b-e, when V_(m) achieves the critical value V_(mc),membrane dielectric breakdown results in the formation of pores in thecell membrane 52, some of which may reseal upon removal of theexternally imposed electric field. FIG. 1b illustrates the condition ofa section of cell membrane 52 from near one pole of the cell with noexternal applied electric field (E=0). When an external field is appliedthat is below the critical field strength required for poration (E<E_(c)FIG. 1c) there is a separation of charge across the membrane 52 and aresulting transmembrane potential V_(m) but no pore formation. Thissituation for the condition E=E_(c), is shown in FIG. 1d, where E is thestrength of the externally applied electric field and, from Eq. 3:

E _(c)=4V _(mc)/3d  (7)

which is the critical electric field strength for spherical cells thatdefines the onset of membrane pore 58 formation for a specific cell sizeand critical transmembrane potential. When V_(m) is less than V_(mc)(i.e., when E<E_(c) in FIG. 1c), pore formation, or at leastirreversible pore formation, does not occur. As V_(m) is increasedbeyond V_(mc) (i.e., when E>>E_(c) in FIG. 1e), membrane pores 59 becomemore numerous, larger, and irreversible. Thus, application ofsufficiently strong electric fields, or PEFs to cellular suspensions canresult in the inactivation of cells by the formation of irreversiblepores which can destroy the function of the semipermeable cell membrane52. As noted earlier, the proper function of a cell's semipermeablemembrane is required to control a cell's intracellular environment and,therefore, to maintain its viability. The critical applied electricfield, E_(c), necessary to form irreversible pores is thus, as isapparent from Eq. 6, directly proportional to the critical transmembranepotential V_(mc), and thus membrane thickness, and inverselyproportional to cell diameter d for spherical cells.

PEF Parameters for Cell Purging and Cell Isolation

As noted above, the lethal effect of pulsed electric fields onbiological cells is caused by irreversible electroporation of theirsemipermeable membranes or reversible poration followed by a subsequenttreatment to prevent membrane repair or accelerate colloidal osmoticlysis and thereby cause cell inactivation. An electric field appliedacross the cell electrically polarizes the cell membrane causing chargeseparation and build up of a transmembrane potential. The criticaltransmembrane potential required for membrane poration will be afunction of the nature and thickness of the membrane, as previouslymentioned, and must be determined experimentally for any given system;however, as previously discussed and illustrated by the data in Table 1,for a wide variety of biological cells the critical transmembranepotential associated with an externally applied field is approximatelyV_(mc)=1 Volt. The pores resulting in the membrane structure of the celldue to exposure to field strengths above the critical value can, incertain cases, irreversibly increase cell membrane permeability leadingto cell death. The dielectric membrane breakdown concept of cellinactivation is illustrated in FIGS. 1b-e.

Electric field strength, total exposure time, and pulse duration, forPEFs, can be selected to preferentially inactivate biological cells in asuspension which are more susceptible to electric fields due, forexample, to their having one or more or a combination of the followingproperties with respect to other cells in the suspension: a largeraverage size; a thinner effective dielectric membrane thickness; a morespherical shape, etc. Of particular importance for many biologicalsamples, especially those having cells with similar shapes, such asroughly spherical, and similar dielectric membrane thickness, isselective inactivation of cells based on a difference in characteristicsize. Typically, the threshold electric field required for cellinactivation is inversely proportional to the characteristic size of thecell, i.e., from Eq. 7, E_(c)(d)=4 V_(mc)/3d, where V_(mc)≅1 Volt is thecritical transmembrane potential for the onset of irreversible poreformation for a wide variety of cell types and d is the diameter orcharacteristic size of the cell. If the undesirable cells are larger indiameter than the desirable cells, then the pulsed electric field methodcan be used to selectively inactivate the larger cells. By operating atelectric field strengths just below the characteristic electroporationthreshold for inactivation of the desirable cells, yet above thecharacteristic electroporation threshold for the undesirable cells, asubstantial fraction of the undesirable cells can be preferentiallyinactivated while leaving a substantial fraction of the desirable cells(primitive stem cells for example) essentially unaltered and stillviable. To further illustrate the utility of the concept, a specificexample related to cell isolation from hematopoietic cell suspensionswill be illustrated. Table 2 lists the types of blood cells thattypically will be present in bone marrow specimens during tumor cellpurging and stem cell isolation processing. The cell diameters, relativeabundance, and projected threshold electric field strengths (E_(c) ascalculated from Eq. 7 assuming V_(mc)≅1 volt) for the onset of membranedamage are also provided in the table. Similar cell sizes as thoselisted would be expected for hematopoietic cells derived from mobilizedperipheral blood, umbilical cord blood and fetal liver tissue, althoughthe relative abundance of each may differ. Table 2 clearly shows thatthe electric field damage threshold for stem cells can be significantlygreater than for the other leukocytes present in bone marrow specimens.Furthermore, the electric field threshold for stem cells can be morethan a factor of two greater than for breast cancer cells. Since, aswill be discussed below, the fraction of cells inactivated by an appliedelectric field scales exponentially with electric field strength(Hülsheger 1983) the factor of two difference in the criticalelectroporation threshold should allow essentially complete inactivationof breast cancer cells with preservation of the viability of the cellscrucial for autologous transplantation (stem cells).

TABLE 2 Electric field damage thresholds for leukocytes and stem cells.Projected Electric Characteristic Relative Field Damage Size AbundanceThreshold Cell Type (μm) (%) (kV/cm) Stem  6^(a,b)  0.001^(a) 2.2Lymphocyte (resting)  7^(b  ) 21^(c )   1.9 Lymphocyte (active) 12^(d) n/a 1.1 Neutrophil 12^(d)  73^(c )   1.1 Eosinophil 13^(d)   4^(c )  1.0 Basophil 15^(d)   0.1^(c)  0.9 Monocyte 15^(d)   2^(c)    0.9 BreastCancer >15^(e)    n/a <0.9 ^(a)Berardi AC, et al: Functional isolationand characterization of human hematopoietic stem cells. Science, 267:104-108,1995. ^(b)Zipori D, et al: Introduction of Interleukin-3 geneinto stromal cells from the bone marrow alters hematopoieticdifferentiation but does not modify stem cell renewal. Blood71:586,1988. ^(c)Jandl JH, Blood: Textbook of Hematology, Little, Brownand Company, Boston/Toronto, 1987. ^(d)Henry JB: Clinical Diagnosis andManagement by Laboratory Methods, 16th Ed., W.B. Saunders Company,Philadelphia, PA, Vol. 1, 1979. ^(e)from observations by inventors

Another feature which, in the present example, further can enhance theability to perform preferential electric field isolation of stem cellsand/or to purge relatively large tumor cells, such as breast cancercells, involves the quiescent nature of stem cells. As discussed inBerardi, et al., stem cells are quiescent and are unaffected by ananti-metabolite treatment, whereas rapidly proliferating cells areinactivated by an anti-metabolite treatment. A similar phenomenon hasbeen observed (Hülsheger 1983) with PEF inactivation of Escherichia coli(E. coli). The observations of Hülsheger 1983 indicate that thestationary growth phase E. coli cells (quiescent cells) are much lessvulnerable to the lethal effects of PEF's than are the larger, rapidlydividing E. coli cells that are in the logarithmic growth phase. Basedon these considerations, it is expected that stem cells, due to theirquiescent nature and smaller size, will be much less vulnerable to thelethal effects of electric fields and PEFs and that electric fieldstrength can be used to preferentially inactivate a substantial fractionof non-stem cell leukocytes and tumor cells while leaving a substantialfraction of the stem cells unharmed. Thus, it is expected that theinventive methods will be an effective approach for purging tumor cellsfrom autologous transplant tissue. Similarly, the inventive methods maybe applied to other cell suspensions or suspensions on non-cell discreteobjects having differences in characteristic size between subpopulationsin order to selectively isolate or inactivate selected subpopulations.

In addition to performing a selective cell isolation or inactivation onthe basis of a difference in characteristic cell size by selecting anappropriate applied electric field strength, the method can also beemployed to select cells that can be similar in size based on adifference in dielectric membrane breakdown, voltage, for example, dueto a difference in effective membrane thickness. For example, a varietyof cells, such as some epithelial cells and cancer cells, can have alayer of mucopolysaccharide coating associated with their plasmamembrane which may increase the effective thickness of the membrane andmake the cells less susceptible to an applied electric field than wouldbe predicted by Eq. 7 with V_(mc) assumed to be 1 Volt. In fact,assuming that the critical electric field imparted to the membranerequired for poration, E_(mc), is similar for the cells present in thesuspension, Eq. 6 indicates that the critical transmembrane potentialV_(mc) will be directly proportional to the effective thickness of thedielectric layer, and, therefore, from Eq. 7, the critical appliedelectric field strength, E_(c), for poration will also be directlyproportional to the effective membrane thickness. Thus, an appliedelectric field strength may be chosen that is sufficient to inactivate asubstantial fraction of cells having an effective membrane thicknessbelow a certain predetermined threshold without inactivating asubstantial fraction of the cells having an effective membrane thicknessabove the threshold.

Although the threshold electric fields for the cells comprisingharvested human bone marrow, as exemplified above, were theoreticallyestimated based on their size (see Table 1), the critical thresholdelectric fields for the cells listed in Table 1 have been previouslymeasured. In addition to the importance of the magnitude of the appliedelectric field strength, total exposure time of the cells to theelectric field is also an important parameter in determining the degreeof inactivation of a given population of cells. In general, for cellsthat are selectively inactivated by electric fields on the basis of cellsize, the electric field strength determines the size below which cellsare preserved, and total electric field exposure time determines therelative reduction in cells having sizes above the critical size.Experiments in the prior art have been conducted over a wide range ofpulsed electric field strengths and number of applied pulses and haveled to an empirical model developed by Hülsheger 1983 hereinincorporated by reference, for the surviving fraction of cells, s,following electric field treatment, as a function of the peak appliedelectric field strength, E, and the total time the cells are exposed tothe electric field, t. The time t in the following model sums theon-time of the electric field over the total number of pulses, so thatt=N_(p)τ_(p), where N_(p) is the number of applied pulses and τ_(p) isthe time duration of each pulse over which E≧E_(c). Hülsheger 1983demonstrated that bacterial cell surviving fraction can be roughlymodeled by an empirical expression that is a power law function of timeand an exponential function of electric field strength. Equation 8provides a variant of Hülsheger's rough model that behaves correctly asthe exposure time approaches zero for E>E_(c). $\begin{matrix}{s = \left( {\frac{t}{t_{c}} + 1} \right)^{\frac{({E - E_{c}})}{k}}} & (8)\end{matrix}$

where, s is surviving fraction (ranging from 0→1), E_(c), is thethreshold value of the electric field strength for membrane breakdown,t_(c) is an exposure time normalization constant, and k is an electricfield normalization constant. E_(c), t_(c) and k can be empiricallydetermined for a given cell suspension by fitting Eq. 8, to data takenrelating fractional inactivation as a function of exposure time andapplied electric field strength using any suitable regression analysisapparent to one of skill in the art.

The equation for surviving fraction s can be utilized as a tool toanalyze data collected for the surviving fraction of a particular cellpopulation vs. applied electric field strength and exposure time,generated for a given cell suspension, and as a guide for selecting thefield strength and duration required to achieve a desired survivalfraction for cells which are to be porated and inactivated. Byprescribing appropriate values for the electric field strength and totalexposure time, required reductions in populations of cells ofelectroporation threshold below a critical value, for example havingsizes larger than a critical diameter, can be achieved. Eq 8 indicatesthat s is a strong exponential function of electric field strength E anda weaker power law function of total electric field exposure time t.

Thus, isolation and inactivation of cells or discrete objects by sizedifferences according to the present invention proceeds by selecting anappropriate applied electric field strength E_(c) so that cells of sizegreater than (from Eq. 7), d=4 V_(mc)/3 E_(c) will be inactivated andthen applying an appropriate number of electric field pulses and/ortotal electric field exposure time to reduce the viable fraction ofcells above the critical size to the desired level.

Table 3 presents the results of cell inactivation experiments assembledby Castro on a variety of microorganisms using Hülsheger's original formof Eq. 8, which is obtained by removing the factor “+1”in Eq. 8(Hülsheger 1983). The threshold electric field E_(c) derived fromlethality measurements for E. coli in the logarithmic growth phase isshown in Table 3 to be 0.7 kV/cm, while the threshold field for E. coliin the stationary phase is more than 10 times higher, i.e., 8.3 kV/cm.The lower threshold electric fields required to irreparably porate themembranes of growing cells are related to the fact that growing cellsare larger and must take in nutrients from the external environment,making them more susceptible to electric fields.

TABLE 3 Experimental conditions, values, and confidence limits for modelparameters. E t E_(c) t_(c) k Microorganism (kV/cm) (ms) (kV/cm) (ms)(kV/cm) E. coli (4 hr)  4-20 0.7-1.1 0.7 ± 3.1  11 ± 9.6 8.1 ± 1.8 E.coli (30 hr) 10-20 0.7-1.1 8.3 ± 0.3  18 ± 5.7 6.3 ± 1.0 K. pneumoniae 8-20 0.7-1.1 7.2 ± 2.0 29 ± 16 6.6 ± 1.4 P. aeruginosa  8-20 0.7-1.16.0 ± 0.4  35 ± 6.1 6.3 ± 1.1 S. aureus 14-20 0.7-1.1  13 ± 0.9 58 ± 172.6 ± 0.7 L. monocytogenes I 12-20 0.7-1.1  10 ± 2.6 63 ± 12 6.5 ± 2.5C. albicans 10-20 0.7-1.1 8.4 ± 7.5 110 ± 33  2.2 ± 0.9

One embodiment of the invention, involving cancer cell purging and stemcell isolation by applied electric fields such as pulsed electricfields, is based on the observation that non-stem-cells are typicallylarger in size than stem cells; therefore, Eq. 7 and the observationthat V_(mc) is typically about 1 Volt for a wide variety of cell typesimplies that an electric field strength, E_(c), can be selected thatwill inactivate a substantial fraction of cells larger than the stemcell, including contaminating tumor cells. After selecting anappropriate predetermined critical electric field strength (which can beapproximately E=2-2.2 kV/cm to preserve stem cell viability), totalelectric field exposure time, t, can then be selected with guidance fromEq. 8, and routine experimental optimization, in order to achieve thedesired reduction in the unwanted, non-stem-cell populations, mostimportantly, tumor cell populations. Therefore, electric fieldconditions can be determined that lead to effective tumor cellinactivation and stem cell preservation, which is crucial for effectivetransplant tissue purging.

Unlike stem cell isolation and purging strategies based onanti-metabolites, mechanical cell sorting, or antibody bindingstrategies, the present invention has the potential to isolate stemcells without damage and without mutation of the basic genetic molecules(DNA/RNA) within the cell. Most importantly, the genetic material isshielded from the pulsed electric fields by the conductivity of the cellnucleus and cytoplasm. Furthermore, at the electric field strengths ofinterest for isolating stem cells (about 1-3 kV/cm), the potentialdeveloped across critical bonds in these complex RNA/DNA molecules isgenerally not sufficient to break these bonds. Hence, stem cellisolation with pulsed electric fields should not cause undo damage tothe genetic material within the cells. An additional advantage of usingthe electric-field-based tumor cell purging, stem cell isolationstrategy is centered on the fact that toxic or potentially activatingagents, such as anti-metabolites or exogenous antibodies, are typicallynot placed in physical contact with the stem cells.

As previously mentioned, because of difficulties in applying acontinuous potential across electrodes without arcing or discharge,electrochemical reactions, and excessive heat generation, the appliedelectric field is preferably supplied to the suspension as a series ofshort pulses, i.e. as a PEF. The maximum electric field pulse durationis typically limited by electric breakdown due to arcing betweenelectrodes in the treatment volume and by single pulse heating effects.The pulse repetition rate is limited by the maximum temperature risethat can be sustained without causing undo damage to the sample. Theminimum allowed pulse duration should be greater than the time constantat which the dielectric membrane charges in response to the electricfield, as will be discussed in greater detail herein.

Thus far, the effect of applied electric field strength and exposuretime on the performance of the inventive methods have been discussed indetail. In addition, as mentioned earlier, a variety of other parametersrelated to the PEF, suspension, pulsing medium (fluid carrier), andother processing steps can affect the performance of the inventivemethod and should be considered when developing an effective isolationor inactivation protocol. What follows is a description of a number ofwhat are believed are important factors related to performance.Throughout the description, reference will be made to one embodiment oftumor cell purging and stem cell isolation from suspensions ofhematopoietic cells in order to illustrate the concepts with a concreteexample. It should be understood that the particular example chosen ispurely exemplary, and the methods may be practiced on a wide variety ofsamples for a wide variety of desired applications. Table 4 belowsummarizes some of the more important parameters (column 2) that caninfluence PEF performance, particularly as related to tumor cell purgingand stem cell isolation, along with contemplated preferred ranges(column 3) of some chosen parameters for tumor cell purging and stemcell isolation. The table will serve as an outline for the discussion tofollow. The “PEF” group includes parameters related to the nature of theapplied electric field. The “Pulsing Medium” group includes parametersrelated to the properties of the suspension. The “Post Processing” groupdiscusses optional treatments subsequent to electric field exposure thatcan, in some cases, enhance performance, and the “Heat Transfer” groupincludes parameters related to the heating effects of the applied PEFs.

TABLE 4 Parameters influencing PEF tumor cell purging and stem cellisolation efficacy. Group Parameter Description Range PEF — Electricfield pulse shape “Rectangular” E Electric field strength 0.5-5 kV/cm tTotal electric field exposure <10 ms time τ_(p) Electric field pulseduration 2-20 μs Pulsing η_(ps) Initial leukocyte 106-108 cells/mlMedium concentration μ_(ps) Pulsing medium ionic 0.015-0.15 M KClstrength equivalent γ_(ps) Pulsing medium osmolality ≦300 mOsm/kg-water— Agents for cell size modification — Agents for dielectric mem- branebreakdown voltage modification Post μ_(ls) Inactivation medium ionic0.15 M KCl equiva- Processing strength and composition lent Cap⁺⁺ τ_(ls)Inactivation medium residence time — Collection protocol Gradientdensity centrifugation Heat F_(p) Electric field pulse rate Apparatus &pulsing Transfer medium dependent T_(ps) Pulsing medium temperature5-41° C.

For embodiments involving PEFs, a substantially rectangular electricfield pulse shape is preferred for achieving optimum size selectivity.Rectangular pulses are those that have rise and fall times that areshort compared to the pulse duration, and preferably shorter than thecharging time scale of the dielectric membrane of the cells or objectsto be inactivated (typically rise and fall times are≦0.5 μs for mostapplications of interest in the present invention), have essentially noovershoot during the rise- and fall-time transients, and have asubstantially constant electric field strength between the rise and falltransients; preferably the difference in the maximum and minimum valuesin the substantially constant field strength region is less than 3%.Non-rectangular-shaped pulses, such as half-sinewave shaped orexponentially decaying pulses may be employed for some embodiments butwill not provide as clearly defined an electric field strength, whichcan broaden the electroporation threshold demarcation line betweenuninactivated and PEF inactivated cells, thereby degrading theselectivity, resolution, and efficiency of the PEF cell inactivationmethod.

The range of electric field strengths given in Table 4 is based on thecritical electric field strengths given in Table 2 and represents areasonable range to employ for optimization trials involving theinactivation of specific cell types listed in Table 2 as a function ofelectric field strength and total exposure time. With this electricfield range, the range of inactivation that can be expected would rangefrom no lethal effects on any cells to essentially total inactivation ofall cell types. Based on Eq. 8, total electric field exposure time t canbe used to achieve a desired reduction in the number of viable unwantedcells. The maximum total electric field exposure time typically will beless than about 10 ms for essentially complete inactivation of tumorcell populations.

For embodiments involving PEFs, the inactivation of biological cellsinvolves multiple steps which are dependent on the duration of theindividual electric field pulses τ_(p). Early in the pulse, the membraneof the cell charges, thereby producing an elevated transmembranepotential. The membrane charging time scale τ_(m) for spherical cells isgiven by (Lynch P T and Davey M R, Electrical Manipulation of Cells,Chapman and Hall, pp. 18-20, 1996.; and Tessié J and Tsong TY, Electricfield induced transient pores in phospholipid bilayer vesicles,Biochemistry 20:(6) 1548-1554, 1981.):

τ_(m)½c _(m) d(ρ_(c)+ρ_(ps)/2),  (9)

where c_(m) is the membrane specific capacitance (typically˜1 μf/cm² forbiological cell membranes (see Schanne OF and P. Cerreti ER, ImpedanceMeasurements in Biological Cells, John Wiley and Sons, New York, p. 331,1978.)), d is cell diameter, ρ_(c) is the resistivity of theintracellular fluid (typically, for cytosol˜100 Ω-cm), and ρ_(ps) is theresistivity of the medium supporting the cells during PEF treatment(typical range of about 70-500 Ω-cm depending on the ionic strength ofthe solution). For a typical cell as exemplified by a hematopoieticcell, the maximum charging time constant will be on the order of about0.5 μs. In preferred embodiments, in order to achieve thorough membranecharging, the duration of the portion of the electric field pulsesupplying an electric field strength greater than the critical fieldstrength, E_(c), to porate the cell, for example the flat-topped portionof a rectangular field pulse, Should be at least three to four times themembrane charging time constant τ_(m), (i.e., for τ_(m)˜0.4 μs,τ_(p)>1.6 μs).

After membrane charging is complete, pore formation begins and transportof ionic species between the inside of the cell and the pulsing mediumtakes place. It is known (Kinosita K and Tsong TY, Voltage-induced poreformation and hemolysis of human erythrocytes, Biochim et BiophysActa.471:227-242, 1977.) (hereinafter “Kinsota 1977”) that ion transportprocesses can enhance PEF cell lysis by disrupting the osmotic balanceacross the cell membrane, which, in turn, can lead to cell swelling andirreversible lysis due to the uptake of water. It is also known(Kinosita 1977) that the efficacy of inducing irreversible cell lysis byPEFs decreases for human erythrocytes when the electric field pulselength is decreased below about 10 μs and improves as the pulse lengthis increased above 10 μs. This phenomenon probably reflects the need foran electric field pulse duration that is sufficient to allow extensivepore development and time for ion species transport. Based on theseobservations, reasonable pulse durations for cells having a size rangeof between about 6 μm and 20 μm, typical for hematopoietic cells, canrange from about a τ_(p)=2 μs to about so τ_(p) =20 μs.

PEF cell inactivation and isolation efficacy can also be affected by thetotal concentration of cells in the PEF treatment volume. The effect oftotal concentration can be understood by comparing the total electriccharge (Q_(m)) required to charge the membranes of all of the cells inthe treatment volume to a transmembrane potential of V_(m)=1 Volt, withthe charge (Q_(p)) actually supplied to the test volume by the electricfield pulse. Q_(m) can be expressed as:

Q_(m)=πν_(TV)η_(c) d ² c _(m) V _(mc)/4,  (10)

where ν_(TV) is the volume of the PEF treatment cell (cm³), η_(c) is theconcentration of cells in the PEF treatment volume (cells/cm³), d is theaverage diameter of the cells in the PEF treatment volume (assume forpurposes of illustration˜10 μm), c_(m) is membrane specific capacitance(assume for purposes of illustration˜1 μf/cm²), and V_(mc) is thecritical transmembrane potential for the onset of irreversible poreformation (assume for purposes of illustration that V_(mc)26 1 Volt).Note that Q_(m) is proportional to the cell concentration, η_(c), Q_(p)can be expressed as:

 Q_(p)=τ_(p)ν_(TV) E/ρ _(ps) w,  (11)

where τ_(p) is the electric field pulse length (sec), ν_(TV) is the PEFtreatment volume (cm³), E is the electric field strength (V/cm), ρ_(ps)is the resistivity of the medium supporting the cells (typically about70-500 Ω-cm) in the PEF treatment volume, and w is the separationdistance between the electrodes in the PEF treatment. Note that Q_(p) isproportional to the electric field pulse length, τ_(p), and inverselyproportional to the pulsing medium resistivity, ρ_(ps). If the ratioQ_(m)/(Q_(m)+Q_(p)) is not small, then cell concentration effects candegrade PEF cell inactivation efficacy by significantly increasing theeffective charging time scale, τ_(m) of the cells in the treatmentvolume, i.e., a significant amount of the total charge supplied to thetreatment volume is required to simply charge the cells. In fact, asthis ratio approaches unity, the cell membranes approach the situationwhere they have just achieved complete charging by the end of theelectric field pulse, so there would be essentially no additional timefor pore development and transport of ionic species between the cell andthe pulsing medium to take place. For best performance, the ratioQ_(m)/(Q_(m)+Q_(p)) is preferably within the range of about0.0004≦Q_(m)/(Q_(m)+Q_(p))≦0.74. The ratio Q_(m)/(Q_(m)+Q_(p)) can bekept constant as the cell concentration (η) is increased by increasingthe pulse length (τ_(p)) proportionally and/or by appropriately reducingthe pulsing medium resistivity (ρ_(ps)), for example by increasing theionic strength. The upper limit of cell concentration given in Table 4(η_(c)=10⁸ cells/ml) has significance relative to a clinical PEF tumorcell purging system embodiment. For example, one liter of bone marrow,which is the approximate volume harvested for autologous transplants,contains approximately 10¹⁰ mononuclear cells, which must be purgedbefore transplantation. If PEF conditions can be defined that providehigh PEF tumor cell inactivation efficacy for a treatment volume cellconcentration of 10⁸ cells/ml, then a 100 ml PEF treatment volume, or aflow-through system able to process 100 ml of cell suspension, can beused to process the entire bone marrow specimen, such a system isreasonable both in terms of system size and electric field pulse energyrequirements.

In addition to the nature of the applied PEF, the medium within whichcells are suspended can be a significant factor in the performance andefficacy of the invention. The pulsing medium, in which biological cellsare suspended, in some embodiments can have an osmolality that preservesthe osmotic balance between the intracellular and intercellular fluids,in other words isotonic, where isotonic defines a solution with anosmotic strength (osmolality) similar to that of the suspended cells sothat the cells do not undergo substantial osmotic pressure-driven cellvolume regulation (˜300 mOsm/kg-water for many mammalian cells, such ashematopoietic cells). If the osmotic strength of the pulsing mediumdiffers substantially from that of the cytosol of the cell, the cell canundergo changes in cell volume, such as shrinkage or swelling that canbe detrimental to cell viability and the performance of the inventivemethod, especially for isolations based on characteristic cell size. Insome embodiments however, it can be desirable to suspend a cellsuspension in a somewhat hypotonic medium either prior to pulsing, withsubsequent pulsing performed in isotonic medium, or during the PEFtreatment itself. In these embodiments, the suspending medium shouldhave an ionic strength selected to enable at least one cell type in thesuspension to undergo osmotic swelling, while not being low enough tocause rupture or a substantial loss of viability to target cells withinthe time frame of the treatment. Pre-treatment, or PEF treatment using ahypotonic suspending fluid can potentially improve performance forcertain cell isolations by causing cells to become larger and morespherical in shape, and thus potentially more sensitive to the effectsof an applied electric field. Treating cell suspensions that arecharacterized by cells having a more uniform spherical shape can alsoimprove performance by reducing the effects of cell orientation withinthe electric field on poration.

The ionic strength of the pulsing medium can be altered, whilepreserving the desired osmolality, by combining a solution comprisingone or more solubilized electrolytes having a desired osmolality (e.g.an isotonic saline solution) with a solution comprising one or moresolubilized non-electrolytes having a desired osmolality (e.g. anisotonic sucrose solution). There are several reasons why, for someembodiments, it can be desirable to reduce the ionic strength of thepulsing medium from standard physiological conditions (e.g. equivalentto a 0.15 M NaCl aqueous solution for many mammalian cells, such ashematopoietic cells). One reason is that it has been shown (Kinosita1977) that PEF treatment using a pulsing solution having abelow-physiological ionic strength followed by resuspension ofPEF-treated cells in a standard physiological ionic strength solutioncan enhance cell destruction by colloidal osmotic lysis and result in amore rapid and extensive irreparable lysis of erythrocytes (red bloodcells). This phenomenon has also been observed in the context of thepresent invention with leukocytes (white blood cells), where the PEFporated cells were subsequently reduced to small cell fragments by PEFtreatment in low ionic strength medium (e.g. 10 v % PBS, 90 v % isotonicsucrose) followed by exposure to a physiological strength medium (e.g.,isotonic PBS or Iscove's Modified Dulbecco's Medium (IMDM)). Forembodiments of the inventive method utilizing a relatively lower ionicstrength pulsing medium followed by exposure to a relatively higherionic strength medium, the particular ionic strengths chosen for thepulse and post-treatment medium, and the exposure time of thePEF-treated cells in the post-treatment buffer will be selected based onroutine experimentation with the particular cell suspension of interestto determine the conditions that yield the highest levels ofinactivation of undesired cells with the best preservation of theviability of desired cells. Typically, the post-treatment medium willhave an ionic strength similar to the physiological ionic strength ofthe cells in the treated suspension and the pulse medium will have anionic strength ranging between 10% and 90% that of the post-treatmentmedium. The exposure time of the cells to the post-treatment medium canvary from a few seconds to an essentially indefinite period. It isimportant, however, that sufficient time be allowed for adequatediffusion and colloidal osmotic lysis to take place. In addition to, orinstead of, having an ionic strength that is higher than that of thepulsing medium, the post-treatment medium may for some embodiments, havea higher osmolality than the pulsing medium and/or contain an agent thatcauses or enhances irreparable lysis of porated cells, such as, in somepreferred embodiments, calcium ions. For some embodiments including apost-treatment step after PEF treatment designed to enhance or causeirreparable cell lysis, the cells porated by PEF exposure undergoirreversible poration of the cell membrane during PEF treatment, andpost-treatment, as described, functions to irreparably lyse the cellsthat have already been inactivated by the PEF exposure. In otherembodiments, exposure to the PEF porates, but does not necessarilyirreversibly porate, the membranes of the cells to be inactivated, withinactivation and/or irreparable lysis occurring during a post-treatmentinactivation step.

For some embodiments, it may be desirable to remove any inactivatedcells, lysed cells, and cellular debris from PEF treated specimens.Small cell fragments and debris generated by cell rupture duringirreparable cell lysis can be separated from the viable cells using avariety of techniques known in the art, for example single- ormulti-gradient centrifugation techniques. For embodiments where PEFs orpost-treatment can fragment affected cells, it is possible to separateviable cells from cellular debris using standard gradient densitycentrifugation techniques. For example, Ficoll-Paque gradient densitycentrifugation, which is a single gradient separation scheme can beused. Multi-gradient centrifugation can also be used for otherapplications as apparent to one of skill in the art. For embodimentswhere inactivated cells also undergo irreparable cell lysis,characterized by cell rupture, it can also be advantageous to add to thesuspending medium an agent that is able to degrade cellular debris. Avariety of such agents apparent to one of skill in the art can beemployed to reduce the cellular debris to its molecular components.Particularly preferred are enzymatic agents, and especially preferred isDNase to breakdown DNA dispersed in the pulsing media, and trypsindigestion to breakdown cell membrane and proteinaceous material. Suchagents may be present during the PEF treatment, or alternatively, may beadded subsequent to PEF treatment.

Other advantages to reducing the ionic strength of the pulsing mediumfor some embodiments are related to the increased resistivity andreduced conductivity of pulsing solutions having a reduced ionicstrength. The power density W_(p) (J/cm³) and total charge density Q(Coulomb/cm³) input into the PEF treatment volume are both functions ofthe resistivity of the pulsing medium:

W _(p) E ²τ_(p)/ρ_(ps)  (12)

Q=Eτ _(p)/ρ_(ps)  (13)

where E is the magnitude of the applied electric field, τ_(p) is thepulse duration, and ρ_(ps) is the resistivity of the pulsing medium. Theresistivity of the pulsing medium varies inversely with ionic strength.Thus, according to Eq. 12, energy and power requirements of a PEFtreatment cell can be reduced by a factor of ten, for example, by usinga mixture of 10% by volume aqueous isotonic phosphate buffered salineand 90% by volume aqueous isotonic buffered sucrose as a pulsing mediuminstead of a standard physiological ionic strength medium. Additionally,reducing the ionic strength of the pulsing medium increases theresistance of the PEF treatment volume, which, for a given electricfield strength and pulse duration, reduces the electron charge driventhrough the PEF treatment volume as shown by Eq. 13. Undesirableelectrochemical reactions, such as free radical production, aretypically proportional to the charge driven through the PEF treatmentvolume. Thus, reducing the ionic strength of the pulsing medium canproportionally decrease the production of free radicals, or otherundesirable by-products of electrochemical reactions. Reduction ofelectrochemical reactions occurring in the PEF treatment volume duringtreatment can also reduce the pH swing of the pulsing medium duringpulsing and also reduce the production of chlorine and hydrogen bubbles.Formation of bubbles during treatment is very undesirable, since thepresence of bubbles can lead to non-uniform field distribution withinthe treatment volume and locally elevated electric field intensities,which can significantly reduce the size selectivity of the inventivecell isolation methods. In general, it is desirable to maintain the pHof the pulsing medium relatively constant during PEF treatment andwithin a range that is not detrimental to the viability of the cellsbeing treated (typically about pH 7-7.6). A variety of buffer systemsapparent to one of skill in the art for use in this range that aresufficiently non-toxic to cells can be employed including, but notlimited to phosphate buffers, BES, MOPS, TES, HEPES, DIPSO, and TAPSO.Preferred for embodiments where precise pH control is especiallyimportant, are buffer systems including one or more strong organicbuffers such as those referred to as “Good” buffers (Good, N. E., etal., Biochemistry, 5:467(1966); Good, N. E., and Izawa, S., Meth.Enzymol., 24(Part B):53(1972); Ferguson, W. J. and Good, N. E., Anal.Biochem., 104:300(1980)), and especiallyN-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES).

One important consideration in designing and implementing a PEFisolation or inactivation strategy is heat effects and temperature risedue to heat generated within the PEF treatment cell due to the energydeposited into the treatment cell by the electric field. One issue thatdepends heavily on heat transfer effects is whether the total exposuretime of the treated suspension should be achieved with a single pulse orwith a series of pulses. Since many biological cells can benon-selectively inactivated by overheating, which is not a function ofelectroporation threshold, and since a single long electric field pulsecan, without sufficient heat removal, cause a greater amount of heatingof the cells for a given total exposure time, it is preferable, for manyembodiments, to apply the total electric field exposure time as a seriesof pulses, rather than as a single pulse of longer duration. Sincecyclic heating and cooling is particularly destructive for blood cells,pulse duration should be kept short enough to minimize cell and cellsuspension temperature excursions beyond the physiological range. Therate, or frequency, at which electric field pulses may be applied isrelated to the energy deposited in the pulsing medium per electric fieldpulse (Joule heating), the geometric shape and heat transfercharacteristics of the particular PEF treatment volume and the densityand thermal conductivity of the pulsing medium (which collectivelydictate heat removal rates), and the heat capacity of the pulsingmedium. There are two components to the total temperature rise in thePEF treatment volume as a function of the electrical energy input: thetemperature jump ΔT_(j)(° C.) that occurs for each individual electricalpulse; and the steady pulsing temperature rise ΔT_(N)(° C.), which is afunction of the volume and heat transfer characteristics of the PEFtreatment cell. Each time a pulse is applied to the pulsing medium, thetemperature will jump. The magnitude of the temperature jump isproportional to the power density W_(p) given by Eq. 12, and can beexpressed as:

ΔT _(j)(° C.)=τ_(p) E ²/φ_(pm)ρ_(ps) c _(p),  (14)

where ΔT_(j) is the temperature jump (° C.), τ_(p) is the electric fieldpulse length (with a typical range for cell isolations involvinghematopoietic cells of about for example 2-20 μs), E is the electricfield strength (with a typical range for cell isolations involvinghematopoietic cells of about for example 0.5-5 kV/cm), φ_(pm) is thedensity of the pulsing medium (typically˜1 g/cm³), ρ_(ps) is theresistivity of the pulsing suspension (with a typical range for cellisolations involving hematopoietic cells of about for example 70-500Ω-cm), and c_(p) is the specific heat of the pulsing medium(typically˜4.19 J/g° C.). Under the most extreme conditions presented inTable 4, and listed in parenthesis directly above, the maximumtemperature jump per electric field pulse will be ΔT_(j)≈0.86° C. Thesteady temperature rise is a function of the heat input per pulse, thenumber of pulses per unit time (frequency), and the heat transfer rateof the PEF treatment cell tending to remove heat from the treatmentvolume. One dimensional conduction heat transfer considerations may beapplied for the static PEF treatment cell embodiment to determine thesteady pulsing, time average temperature rise at the midplane betweenthe electrodes imparting the electric field to the treatment volumerelative to the temperature of the bounding electrodes. This formulationassumes that convective transport driven by temperature induced densitygradients are of negligible importance. Equation 14a below (see Holman JP. Heat Transfer, 5th Ed., McGraw-Hill, Inc., New York, 1981, p. 35)describes the average temperature rise for one dimensional steadypulsing volumetric heat deposition by PEFs into a treatment volumebounded by two plane electrodes.

ΔT _(Cl,ave) =qw ²/8κ  (14a)

Where q is the volumetric heat deposition rate given by:

q=Fτ _(p) E ²/ρ_(ps)  (14b)

And where w is the separation distance between the electrodes, κ is thethermal conductivity of the pulsing medium, F is the pulse repetitionfrequency, τ_(p) is the electric field pulse length, E is the strengthof the imposed electric field, and ρ

is the resistivity of the pulsing medium.

Equations 14a and 14b indicate that the mid-plane temperature increasesas the square of the electrode separation distance, linearly with pulserepetition frequency, the square of electric field strength, andinversely proportional to the resistivity of the pulsing medium. Sincethe average midplane temperature rise given by Eq.'s 14a and 14b, whichconstitutes the maximum average temperature rise in the PEF treatmentvolume, is inversely proportional to the resistivity of the pulsingmedium, an increase in pulse repetition frequency by a factor of ten canbe realized while maintaining the same midplane temperature rise byincreasing the pulsing medium resistivity by a factor of ten. This canbe accomplished by decreasing the ionic strength of the pulsing mediumto 10% of standard physiological ionic strength by the methods describedpreviously. The temperature jump given by Eq. 14 in response to theapplication of each electric field pulse can be imagined to besuperimposed on the average steady pulsing temperature rise ΔT_(Cl,ave)as a periodic temperature spike with an exponential decay and afrequency corresponding to electric field pulse repetition frequency.

For illustrative purposes, if the electrode spacing is 0.318 cm andusing the most extreme conditions presented in Table 4, which gave amaximum temperature jump per electric field pulse of ΔT=0.86° C., thesteady pulsing temperature rise at the midplane between the twoelectrodes will be ΔT_(Cl,ave)(° C.)=5.16 F.(Hz). Thus, if theelectrodes are maintained at a temperature of 25° C. and one wishes notto exceed a midplane peak temperature no greater than 37° C., then theaverage midplane temperature cannot exceed ΔT_(Cl,ave) =37−25−ΔT _(j),which is ΔT_(Cl,ave)=11.14° C., which constrains the pulse repetitionfrequency to be no greater than F=11.14/5.16=2.16 Hz. If the ionicstrength of the pulsing medium was reduced by a factor of ten, thepulsing medium resistivity would increase by a factor of ten, whichwould allow operating at a pulse repetition frequency of 21.6 Hz forwhich ΔT_(Cl,ave) would remain at 11.14° C.

For illustrative purposes, assuming a worst-case heating situation byneglecting any PEF treatment volume heat removal effects, the totalelectric field exposure time (t=N_(p)τ_(p), where N_(p) is the number ofapplied electric field pulses) that may be applied without exceeding apredefined pulsing medium temperature rise can be expressed as:

t=φ _(pm)ρ_(ps) c _(p) ΔT _(max) /E ²,  (15)

where ΔT_(max) is the maximum predefined allowable temperature rise (°C.). For example, if pulsing is initiated with a PEF treatment cellpulsing medium temperature of 25° C. and we wish not to exceed a finalpulsing medium temperature of 37° C., then the predefined maximumallowable temperature rise is ΔT_(max)=12° C. Based on ΔT_(max), and thestrength of the applied electric field the allowable total electricfield exposure time for a given cell suspension in a PEF treatment cellcan be determined by Eq. 15. Eq. 15 also shows that if a low ionicstrength pulsing medium is used, the allowable electric field exposuretime calculated for a given ΔT_(max) is directly proportional to thedecrease in ionic strength; for example, reducing the ionic strength bya factor of ten would increase the allowable exposure time by about afactor of ten. Furthermore, since under low ionic strength conditions,the energy transferred to the PEF treatment volume is reduced, for agiven desired total exposure time, the pulse frequency may be increasedwithout exceeding the predefined maximum temperature rise, thus allowingfor a more rapid isolation or inactivation. Accordingly, the inventivePEF strategy can be a very rapid approach to cell purging and cellisolation.

For a single- or multi-pass flow-through PEF treatment cell embodimentof the invention, Eq. 15 can be manipulated to a form that describes thetemperature rise of a fluid element of the pulsing medium as ittraverses from the inlet to the exit of the PEF treatment volume. Thisformulation represents an upper bound on the temperature rise since itneglects heat transfer to the bounding electrodes as the fluid elementpasses through the PEF treatment volume. If N_(p) is the number ofelectric field pulses applied during the residence time of the pulsingmedium in the PEF treatment volume and τ_(p) is the duration of each ofthe electric field pulses, then the total electric field exposure timeis t=N_(p)τ_(p) and the resulting temperature rise is given by Eq. 15abelow:

ΔT _(res) =tE ²/φ_(pm)ρ_(ps) c _(p)  (15a)

As illustrated above for the static PEF treatment cell embodiment,reduction of the ionic strength of the pulsing medium, which results inan increase in the resistivity of the pulsing medium, can allow acorresponding increase in the number of pulses that can be appliedduring the residence time of the pulsing medium in the PEF treatmentvolume. Thus, for the flow-through PEF treatment cell embodiment, it canbe beneficial to use the lowest ionic strength pulsing medium allowablein order to maximize the number of pulses that may be applied in asingle pass without exceeding the temperature rise limitations beyondwhich thermal effects impact cell viability. The relationships givenabove provide guidance to the skilled practitioner or selectingreasonable pulse repetition frequencies that are appropriate, for aspecific pulsing medium and electric field pulse intensity and duration,for inactivating selected cells from a cell suspension having a maximumallowable temperature rise.

Another previously mentioned factor that can influence the way in whichan applied electric field interacts with a cell suspension, and theselectivity of an applied electric field at inactivating cells based ona critical electroporation threshold, is the shape and orientation ofcells within the field. This factor is important for any cellinactivation involving non-spherical shaped cells. A problem arises withsuch samples because non-spherical cells, in a given sample, aretypically randomly aligned with respect to the electric field direction.While such a random alignment is not a problem for hematopoietic stemcells or other essentially spherical cells, random orientation canreduce the effectiveness, especially for cells with large aspect ratios,of cell inactivation with applied electric fields. Eq. 1 shows that thetransmembrane voltage, V_(m), that results from an applied electricfield E is directly proportional to the projected length, l, of the cellin the electric field direction. Thus, for cells with large aspectratios, l can be highly variable depending on the orientation of thecell. Since it is typically desirable to apply an essentiallytime-invariant transmembrane voltage for a predetermined length of timein order to obtain more easily predictable and controllable porationresults, it is therefore desirable to align cells that are notsubstantially spherical so that they have a more consistent andpredictable orientation with respect to the electric field direction.For embodiments of the invention where it is desired to align the axesof cylindrical or oval shaped cells to achieve maximum PEF inactivationefficiency, an AC field can be applied across the sample to accomplishthis function. The AC field is preferably selected to provide anessentially uniform oscillating electric field during the PEF treatmentperiod, and has a magnitude selected to be sufficient to align the cellswith their long dimensions parallel to the PEF field direction foroptimum size selectivity by the PEF field, without porating the cells orunduly overheating the pulsing medium. The theoretical treatment of thiscell alignment technique is discussed in detail by Lynch (Lynch P T andDavey M R. Electrical Manipulation of Cells, Chapman and Hall, New York,Chapter 4, 1996), herein incorporated by reference.

Some preferred embodiments of the invention include the use of anapplied electric field for cell inactivation that is a bipolar electricfield. A “bipolar electric field” as used herein refers to an electricfield that is pulsed or otherwise applied to a sample so that theaverage current across the sample over the total treatment time isessentially zero. The use of bipolar electric fields in the context ofthe present invention provides several advantages over non-bipolarfields. When an electric field is applied across a sample, particularlya blood sample, electrochemical reactions can occur which can producefree radicals, other deleterious compounds, and species that can shiftsuspension pH and/or generate bubbles. Such electrochemical effects are,as previously indicated, undesirable. Within the context of the presentinvention, the inventors have found that undesirable electrochemicaleffects can be reduced or eliminated by utilizing a bipolar electricfield pulsed so that the average current across the sample over thetreatment time is essentially zero. Because the application of thebipolar electric field involves essentially equal current flows acrossthe sample for each applied polarity, the reversible electrochemicalreactions induced by the applied electric field component having a firstpolarity, can be substantially reversed by the applied electric fieldcomponent having the opposite polarity, thus yielding a situationcharacterized by no net electrochemical reaction over the treatmenttime.

There are a variety of ways to apply a bipolar electric field to thesample as apparent to one skilled in the electrical engineering arts.For some embodiments, the pulses across the sample are of essentiallyequal magnitude, duration, and number, but alternate pulses are ofopposite polarity, while for other embodiments, the pulse having a firstpolarity may be of greater magnitude but shorter duration while thepulse of the reverse polarity is of lower magnitude and longer duration,so that the total average current flow is essentially zero. In anotherembodiment, an electric field pulse having a first polarity is utilizedtogether with a DC current having an opposite polarity selected so thatthe magnitude and duration of each is selected to yield an essentiallyzero net current in order to achieve no net electrochemical reactionwithin the sample. In yet another embodiment for creating a desiredbipolar electric field, the pulse used to create the PEF field may alsobe utilized to charge a suitable capacitor. When the original PEF pulseterminates, the capacitor then discharges back through the solutioncontaining the cells at a rate determined in part by a resistance in thedischarge path to produce the desired bipolar field.

In addition to reducing undesired electrochemical reactions, bipolarPEFs can provide an additional advantage in the selectiveinactivation/lysing of larger cells. The additional advantage lies inthat the first pulse component having a first polarity results in acharge across the membrane of the cell which remains for some period oftime after the first pulse component terminates. If a second pulsecomponent having an opposite polarity is applied across the cell duringthis time, the voltage across the cell can be effectively doubled for ashort period of time.

This doubling effect is greater for larger cells than for smaller cellsbecause of the larger membrane charging time scale for larger cells (seeEq. 9). This effect can potentially enhance size-selective destructionof the larger cells, thereby enhancing the cell selectivity of theinvention for certain applications.

Temperature may also be utilized to enhance cell inactivation by theinventive methods. In general, biological cells are less capable ofrepairing membrane damage at sub-physiological temperatures. Thisbehavior can be utilized to increase inactivation of cells in responseto an applied electric field. For example, in one embodiment the cellsare subjected to a PEF in a solution that is maintained within aphysiological range of temperatures (for most mammalian cells,approximately 33-39° C.), the porated cells are then resuspended in asolution at a lower temperature, for example room temperature(approximately 25° C.) or lower, but above the freezing temperature ofthe suspension. The lower temperature solution delays repair of poratedmembranes and thus can increase the degree of cell inactivation bycolloidal osmotic lysis.

Alternatively, since cell repair (i.e. the closing of pores in a poratedcell) will not take place when the cell temperature is dropped muchbelow body temperature, the PEF exposure itself can be performed at alower temperature, thereby permitting irreversible poration to occur atan electric field strength that can be lower than that which wouldotherwise be required. In addition, utilizing a lower PEF subjectingtemperature for any given applied electric field strength can improvethe kill rate (i.e. reduce the surviving fraction) from that suggestedby Eq. 8 as determined for the same given field strength but a higherPEF subjecting temperature.

For embodiments of the invention involving applications where it isdesired to selectively inactivate one or more cell types having acharacteristic size greater than a predetermined value whilesimultaneously leaving substantially viable another desired cell type,or group of cell types, having a characteristic size below thepredetermined value, the greater the difference between thepredetermined value of size and the characteristic size of the desiredcells, in general, the easier and more selective the isolation.“Substantially viable” as used herein indicates that at least about 10%of the cells in the population are viable, preferably at least 50%, morepreferably at least 90%, and most preferably at least 95%, whileconversely, “substantially non-viable” as used herein indicates that atleast about 25% of the cells in the population are non-viable,preferably at least 75%, more preferably a least 90%, more preferably atleast 95%, and most preferably essentially all of the cells in thepopulation are non-viable. For applications where the difference incharacteristic size of the desired cell type and the undesired cell typeis small, a variety of strategies can be utilized to improve theperformance of the PEF isolation protocol. One method is to remove thecells that are close in size to the desired cells by performing apreliminary cell separation step that separates cells on some basisother than a difference in size. Depending upon the particularapplication, suitable cell separation methods include but are notlimited to: flow cytometry; affinity cell chromatography; andcentrifugation.

For example, in an application involving the isolation of hematopoieticstem cells from other blood or bone marrow cells, both red blood cells(about 7 μm diameter) and resting lymphocytes (about 7 μm diameter) aredimensionally the closest to the stem cell (about 6 μm diameter).Because the number of red blood cells is much greater than the number ofall of the nucleated cells combined, and because the red cells have ahigher density than the nucleated cells, they are typically separatedfrom a sample by density gradient centrifugation, using for example aFicoll-Paque single gradient having a density of 1.07 g/cm³ (which willalso remove polymorphonuclear leukocytes such as neutrophils), beforePEF application. If the size selectivity of the PEF isolation for aparticular embodiment is not sufficient to select stem cells over theresting lymphocytes with the desired level of purity, the restinglymphocytes, or other small cells, can be removed from the pulsingmedium prior to PEF treatment. This can be achieved, by a variety ofcell separation methods known in the art including CD34 targetedantibody affinity binding techniques that are selective for CD34⁻ cells,such as hematopoietic precursor cells including some stem cells. Analternative is to add an agent to the cell suspension that canpreferentially modify (increase or decrease) the criticalelectroporation threshold of a subset of the cells to make them more orless susceptible to inactivation by electric fields. For example in oneembodiment, the small resting cells, such as resting lymphocytes, may beeliminated by activating the resting lymphocytes using an agent thatforces the resting lymphocytes into their active state so as to achievetheir mature size (typically about 12 μm) where they can be more easilyinactivated without adversely affecting the stem cell population. Avariety of suitable activating agents are known in the art includingprotein agents, such as cytokines, lymphokines, chemokines, anti-cellsurface marker antibodies, and cell receptor antagonists. Other suitablestimulants include lectins, such as Phaseolus vulgaris lectin (PHA), andconcanavalin A. As specific examples, monoclonal anti-CD40 antibody,CD40 ligand, or interleukin-4 (IL-4) can efficiently activate restingB-cells (see Valle A, Zuber C E, Deffrance T, Djsou, et al., Activationof human B lymphocytes through CD40 and Interleukin 4. Eur J Immuno19:1463-1467, 1991. Banchereau J, de Paoli P, Valle A, Garcia E, et al.,Long term human B cell lines dependent on Interleukin 4 and anti-CD40.Science 251:70-72. 1991. both incorporated herein by reference), whileinterleukin-2 (IL-2) and concanavalin A can efficiently activate T-cells(Berger S L, Lymphocytes as resting cells. Methods in Enzymology. (eds)Jakoby W and Pastan I. Academic Press. Vol LVIII:486-494, 1979. Waxman Jand Balkwill. (eds) Interleukin-2. Blackwell Science Publications,Oxford, 1992. both incorporated herein by reference).

Another factor that can affect the performance of selective cellinactivation by electric fields on the the basis of a difference in acharacteristic electroporation threshold based on cell size is aconcurrent difference in the dielectric membrane breakdown voltage, forexample, due to a difference in the effective membrane thickness,between cell types. In such cases, it can be advantageous to add anagent to the cell suspension, prior to or concurrently with electricfield application, that can modify (increase or decrease) the dielectricmembrane breakdown voltage of one or more cell types. For example, forapplications where a larger, undesirable cell type possesses a thickereffective membrane thickness than a smaller desired cell type, aspreviously discussed, the larger cell having the thicker effectivemembrane thickness will typically have a higher critical dielectricmembrane breakdown voltage and thus require a larger electric fieldstrength for inactivation than a comparably sized cell having a thinnermembrane. Depending on the difference in characteristic size andeffective membrane thickness between the two cell types, the ability ofthe electric field to selectively porate the larger cells while notaffecting the smaller cells can be diminished or eliminated. A specificexample of an application where this phenomenon may arise is in the useof PEFs for selectively inactivating certain tumor cells from tissuesamples or hematopoietic cell suspensions. A variety of tumor cells, forexample many epithelial tumor cells, have associated with their plasmamembrane a relatively thick layer of mucopolysaccharide, known as theglycocalyx, that can increase the effective thickness of the membranedielectric layer making the cells less susceptible to the PEF. Forapplications where this phenomenon is an important consideration, e.g.when cell kills are substantially less than would be predicted based oncell size alone, the performance of the PEF method can be improved byremoving the glycocalyx layer prior to subjecting the cell suspension tothe PEF treatment. Agents that can be used for effectively reducing oreliminating the glycocalyx layer on cell membranes include enzymes suchas hyaluronidase, collagenase, pronase, elastase, and trypsin (GruenertD C, Basbaum C B, and Widdicombe J H. Long-term culture of normal andcystic fibrosis cells grown under serum-free conditions, In Vitro Cell.Dev. Biol. 26, 411-418, 1990. Lechner J F, Babcock M S, Marnell M.Narayan K S, and Kaighn M E. Normal human prostate epithelial cellcultures, Methods in Cell Biology, 21B, 195-225, 1980. Stamfer M R.Isolation and growth of human mammary epithelial cells, Journal ofTissue Culture Methods, 9, 107-115, 1985.). Alternatively, an agent thatpreferentially increases the dielectric membrane breakdown voltage of adesired cell type could be used, in addition to or instead of the abovementioned agents, in some embodiments to make the cells less susceptibleto inactivation by electric fields.

In some embodiments, where it is desired to alter the apparent membranebreakdown voltage or characteristic size of one or more subpopulationsof cells in a heterogeneous population of cells, for example when theelectroporation thresholds of desirable and undesirable cellular subsetsare comparable, such alterations can be effected by attaching materialto cellular subsets using antibodies that have immunospecificity for thecells, especially monoclonal antibodies. For example, metallic beadscoated with a monoclonal antibody that can bind the bead to a specificcell surface antigen can produce two distinctly different effectsdepending upon the surface density of the beads attached to the cell. Ifthe surface density of the attached beads is very high, such that analmost continuous layer of metallic beads exists on the surface of thecell, then the resulting metallic structure will behave as a Faradaycage, which will shield the cell from the effects of the imposedelectric fields. If the surface density of the beads on the cells islow, however, then the each bead will behave as an antenna, which canmake the effective size of the cell larger, thereby making the cellsmore susceptible to the lethal effects of the imposed electric fieldsthan implied by the original size of the cell. Thus, agents, such asantibody coated metallic beads, can be used to alter the electroporationthresholds of specific cells, thereby enhancing PEF selection orinactivation characteristics.

A typical stem cell, shown in FIG. 2a, also possess a unique morphologythat can make them less susceptible to poration by electric fields thantheir size would suggest. Morphologically, stem cells are typicallysmall in size (˜6 μm in diameter for hematopoietic stem cells) with afaint halo of cytoplasm 72 between the nuclear sack 74 surrounded bynuclear membrane 73, and outer (plasma) membrane 71. The next largernucleated hematopoietic cells, resting lymphocytes (˜7-8 μm indiameter), typically have a much larger gap between their nuclear andouter membranes. The arrangement of the stem cell's nuclear 73 and outermembranes 71 being separated by a very small distance can cause thenuclear 73 and outer membrane 71 to become electrically coupled and tocharge together as one effective dielectric layer of thicknessapproximately equal to the sum of the thicknesses of the nuclear andouter membranes. Provided the electric field pulse length is smallcompared to the discharge time scale of the nuclear membrane, electricfield strengths considerably greater than the value implied by thediameter of the stem cell and the critical transmembrane voltage, V_(mc)for the outer membrane 71 will be needed to form temporary orirreversible pores in stem cells. Specifically, for PEF pulse durationsless than the characteristic discharge time scale of the nuclearmembrane 73, the critical electric field required for the onset ofporation would be:

E _(coup) ≈E _(c)(t _(om) +t _(nm))/t _(om)  (16)

where E_(coup) is the required electric field strength to porate thecell for electrically coupled nuclear 73 and outer 71 membranes, E_(c)is the critical field strength as calculated from Eq. 7, t_(om) is theeffective thickness of the outer membrane 71, and t_(nm) is theeffective thickness of the nuclear membrane 73.

FIG. 2b illustrates electrically the reasons the nuclear and outermembranes of the stem cell can charge as one membrane when the electricfield pulse duration is small compared to the discharge time scale ofthe nuclear membrane. The poles 81, 82 of the cell are defined as thosetwo points on the surface of the cell which are closest to theelectrodes imposing the electric field. The membrane gap is defined asthe smallest distance between the nuclear and outer membranes. Thepole-to-pole membrane gap resistance 80 is the resistance in themembrane gap between opposite poles of the cell. The membrane gapresistance 77 is the gap resistance between the inner and outermembranes. When the membrane gap is very small, the membrane gapresistance 77 will be much less than pole-to-pole membrane gapresistance 80. This is typically the case for the stem cell. Under theseconditions the nuclear and outer membranes will charge together as onemembrane of thickness approximately equal to the sum of the thicknessesof the nuclear and outer membranes. When the gap is significant, whichwill be the case for most other cells in the blood and immune system,the pole-to-pole membrane gap resistance 80 is less than the membranegap resistance 77, and the nuclear membrane does not participatesignificantly in electric field effects, so that just the outer membranecharges. If R_(pp) is taken as the pole-to-pole membrane gap resistance80 and C_(pp) is the capacitance of the dual membrane system, then thedischarge time scale of the nuclear membrane will be no greater thanτ_(nm)<R_(pp)C_(pp). Provided the electric field pulse is significantlyless than τ_(nm), the nuclear and outer membranes will behaveelectrically as one membrane of thickness equal to the sum of thenuclear and outer membrane thicknesses (see Eq. 16) and during thistime, electric field strengths much greater than implied by the diameterof the stem cell (see Eq. 7) will be required to form irreversiblepores. This effect can enhance the ability to isolate stem cellsutilizing the PEF methods of this invention and can reduce the need toactivate cells that arc close in size to the stem cells in order toincrease the difference in characteristic size.

While the discussion above has been with respect to the poration oflarger cells to isolate or segregate stem cells which typically have asmaller size, there are also applications where it may be desirable toporate the stem cells. Heretofore, an effective technique forelectroporating stem cells has not existed. Poration of stem cells canbe achieved according to the present invention by initially using thePEF method to isolate stem cells, and subjecting the isolated stem cellsto an electric field of appropriate magnitude for porating the stemcells. For example, if the electric field pulse duration, τ_(p), beingutilized is less than τ_(nm), then the appropriate magnitude would bedetermined by employing Eq. 16 in combination with Eq. 7; however, ifthe pulse duration is longer than τ_(nm), then the appropriate magnitudewould be determined by employing Eq. 7. The objective in porating stemcells can be reversible, non-lethal poration or irreversible poration tolyse or kill the cells. One embodiment of the present inventive methodscan enable the temporary, reversible poration of the stem cells toallow, for example, genetic material to enter the stem cells, producinggenetic mutations or recombinations for gene therapy. The inventive stemcell transfection technique may be preferable to many currently employedtechniques, such as using viral transfection, since the PEF porationtechnique can enable the genetic material to enter a larger percentageof the stem cells and can result in a higher survival rate for themutated stem cells. Such mutated stem cells can, for example, beutilized in a variety of gene therapy techniques, for cloning, or toprovide immunity against specific adverse biological agents such asviruses, bacteria, and various toxins.

Since a variety of known methods exist for extracting viable cells frommixtures containing viable/dead cells and cellular debris, selectiveinactivation of cells by the methods provided by the present inventionrepresents a potentially important step toward achieving high purityisolation. PEF cell inactivation can, via post-PEF spontaneous celllysis (colloidal osmotic lysis), transform the PEF inactivated cellsinto ghost membranes and free nuclei dispersed in the cellularsuspension. Single or multiple gradient centrifugation techniques canthen be used to separate the viable cells, and, if desired, any residualintact non-viable cells, from the cellular debris composed primarily ofghost membranes and free nuclei. Viable cells can also be extracted fromthe PEF-treated mixture of viable/dead cells and cellular debris byusing cell sorting, e.g. FACS or flow cytometry, or antibody bindingstrategies that are commercially available for a variety of cell types.Alternatively, if the population of selected viable cells are to beexpanded, there may be no need to remove the post-PEF non-viable cellsand debris, since the expansion process can produce a post-expansionpopulation of viable cells that will render insignificant the relativelysmall numbers of inactivated cells and residual cellular debris, or thedebris will simply decompose during the expansion culture.

FIG. 3 presents a flow chart summarizing the steps of a typical PEF cellisolation strategy according to the invention. It should be reemphasizedthat for any given cell suspension and desired cell isolation orinactivation, the PEF parameter values for optimal performance must beselected based on routine experimentation and optimization with guidancefrom the theoretical development presented previously. The generalprocedures described below may be employed both during screening teststo determine optimal PEF parameters for cell isolation, and duringactual cell selections with predetermined optimal parameters.

Initially, before the beginning of the procedure, PEF operatingparameters (e.g. electric field strength, total exposure time, pulseduration and frequency, etc.) are selected as described previouslybased, in part, upon a difference in a characteristic electroporationthreshold difference between desired and undesired cells (determineschoice of electric field strength) and a desired degree of cellinactivation (determines choice of exposure time). The first step 61 ofthe procedure 60 is to prepare the cell suspension. The cell suspensionis prepared by uniformly dispersing and suspending cells in aphysiologically compatible, conductive medium. In some cases, e.g.blood, the sample to be treated may already be suspended in a suitablemedium, in other cases, e.g. cells from solid tissue or organs, thecells may need to be dispersed and resuspended in a suitable medium. Theviability, concentration and identity of the cells present in thepre-PEF treated suspension can be determined by a variety of methodsknown in the art. Viability, for example, for many cells, such asmammalian cells, can be determined by trypan blue dye exclusion.Concentration may be determined by manual or automated cell countingtechniques, for example manual counting with a hemacytometer, orautomated counting by light scattering techniques. Individual cell typescan be enumerated and marked for further tracking by standardimmunophenotyping techniques known in the art, such as by usingcell-specific dyes or dye-labeled antibodies (e.g. fluorescently labeledantibodies) that have specificity for certain cell surface antigensspecific to certain cell types. The labeled cells can then be quantifiedby standard techniques, for example, fluorescence microscopy or flowcytometry.

The second step 62 of the procedure 60 includes various optionalpre-treatment methods, or pre-PEF cell separations, used to enhance theperformance of the PEF treatment. A variety of such methods werediscussed previously and include: isolation of a subpopulation of cellsby cell separation methods such as centrifugation, for example, densitygradient centrifugation; osmotic cell lysis of red blood cells; cellaffinity chromatography; fluorescence activated cell sorting (FACS);etc. Other treatments that can be employed at this step includetreatments designed to enhance a difference in characteristicelectroporation threshold, such as: hypotonic swelling of the cells;treatment with an agent to remove a glycocalyx layer from one or morecell types to reduce the effective membrane thickness; treating the cellsuspension with an activating agent to increase the characteristic sizeof one or more cell types, etc. For applications involving the isolationof hematopoietic stem cells from blood or bone marrow, typically duringthis step, mononuclear cells are purified from the sample from step 1 byusing a standard Ficoll-Paque density gradient centrifugation technique,followed by ACK (product 10-548, BioWhittaker, Walkersville, Md.) lysisof residual erythrocytes. The mononuclear cells are then washed andresuspended in an appropriate pulsing buffer. Optionally, an activatingagent, such as those mentioned previously, may be added to thesuspension to activate resting lymphocytes, or, if tumor cells having athick glycocalyx layer are present, an enzyme may be added to at leastpartially remove this layer.

The third step 63 of the method 60 involves subjecting the cellsuspension to the pulsed electric field. Electric field strengths anddurations may be selected for this step that are sufficient to causeirreversible poration and inactivation of the porated cells, oralternatively, that are sufficient to reversibly porate but notinactivate porated cells. For some purposes, the PEF treated cellsuspension produced during this step is in a final, usable form;however, for many applications, additional post-PEF steps are requiredor desirable for attaining a final cell suspension.

The fourth step 64 of the method 60 is an optional step performed toinactivate cells that are porated but not inactivated in the previousstep 63. This step 64 is typically employed for embodiments where thePEF parameters chosen in step 63 are sufficient to porate but notinactivate the undesired cells; however, the step may also be employedfor embodiments utilizing PEF conditions selected for inactivation inorder to, for example, increase the total fraction of cells inactivated,speed up the inactivation process, or physically disrupt the structureof the inactivated cells by irreversible lysis. As previously discussed,the methods of this step typically involve resuspending the PEF treatedcells in an inactivation medium, or adding one or more supplementalagents to the PEF medium to create an inactivation medium in situ,designed to accelerate colloidal osmotic lysis, prevent cell membranerepair, or both. As previously discussed, the inactivation medium foruse in this step typically will have one or more of the followingproperties: a higher ionic strength than the pulsing medium; a higherosmolality than the pulsing medium; a lower temperature than the pulsingmedium; or an agent (e.g. Ca⁺⁺) that specifically promotes colloidalosmotic lysis of porated cells.

The fifth step 65 is an optional step performed to remove inactivatedcells and cellular debris from the suspension containing viable PEFisolated cells. This step can be performed by a variety of techniquesapparent to the skilled artisan, including, but not limited tocentrifugation, filtration, and adsorption. In some embodiments,suitable agents may be added in order to break up or reduce cellulardebris, such agents including, for example DNase, trypsin, or otherenzymes. For applications involving hematopoietic cell isolations, afterPEF treatment, the stem cell enriched suspension is typically subjectedto a Ficoll-Paque gradient density centrifugation to remove inactivatedcells and cellular debris.

For selected applications, it may be desirable to further purify,isolate or treat a subpopulation of cells from the PEF treated cellsuspension. Such secondary, or supplemental cell separation or othertreatments comprise an optional sixth step 66. Any of the previouslymentioned cell separation techniques can potentially be employed forthis step. In some cases, the secondary purification may be used tofurther enrich or purify the subpopulation initially isolated by PEFtreatment, while in other cases, the secondary isolation may involveseparating a sub-subpopulation of cells from the PEF isolatedsubpopulation, which sub-subpopulation may not be distinct with respectto a critical electroporation threshold. Such a secondary separation ispotentially useful, for example, for applications where it is desirableto separate a particular type of progenitor cell from a PEF isolatedstem cell suspension. As previously discussed, preferred stem cellsuspensions isolated by PEF can contain a variety of different lineagecommitted colony forming cells in addition to the pluripotent cells. Oneor more of these cell sub-types may be isolated by, for example, FACS byutilizing one or more labeled antibodies with immunospecificity to cellsurface markers present on particular cell sub-types.

The final optional seventh step 67 comprises any treatments that areperformed on or using the PEF treated cell suspensions. Such treatmentscan include, for example, expansion and/or differentiation of theselected cells by in vitro cell culture techniques, transfection orother genetic modification of selected cells, etc. For example forapplications involving the isolation of stem cells, since the fractionof stem cells present in a typical pre-treatment sample is very small(about 1:10⁶ in bone marrow aspirate), depending on the volume of sampleprocessed, the final quantity of isolated stem cells may not besufficient to engraft a patient (typically about 10⁶ CD34⁺ cells/kg bodyweight are required). In such cases, the quantity of stem cells can beamplified through standard stem cell amplification and expansiontechniques (Zandstra, A. J., et al. “Advances in hematopoietic stem cellculture,” Curr Opin Biotechnol 9:146-151(1998)). Cell culture techniquescan also be employed to activate the isolated stem cells todifferentiate along desired lineage paths, thus increasing the number ofcommitted progenitor cells reinfused into a patient. An additionalpost-PEF isolation treatment that can optionally be performed on theisolated stem cells, for example for gene therapy applications, isreversible poration and transfection of the cells with genetic materialusing PEFs to electroporate the stem cells.

In most applications, and especially when screening to determine optimalPEF parameters, it is desirable to characterize the post-PEF treatmentcell suspension with regard to cell quantity, cell viability, and cellidentity in order to evaluate performance. The techniques described forcharacterizing the pre-treatment suspensions can also be employed tocharacterize the post-treatment populations. From comparison of the pre-and post-treatment cell suspensions, yield, selectivity, enrichment, anddepletion determinations can be inferred. The inventors have found thatfor analyzing post-PEF suspensions of hematopoietic cells, an effectiveand convenient method to simultaneously determine cell viability, cellapoptosis, and cell concentration, and cell identity, is to stain thepost-PEF cells obtained after the third step 63 or the fifth step 65above with a combination of propidium iodide, Annexin-V, andfluorescently labeled antibodies specific to CD3, CD14, CD19, CD45,CD34, and CD38 cell surface markers. These cell specific fluorescentlylabeled markers allow enumeration of lymphocytes (CD3+, CD45+),monocytes (CD14+, CD45+), primitive progenitor cells (CD34+), andcertain hematopoietic stem cells (CD34+, CD38−). The viability stainpropidium iodide is a DNA stain. Thus for cells with disruptedmembranes, yet still containing a nucleus, this dye will stainnon-viable cells. Annexin-V, however, stains phosphatidylserine (PS)which migrates from the inside to the outside of the plasma membraneduring normal apoptosis. Thus it is normally used as an indicator ofapoptotic, not yet non-viable cells. Whether or not the PS migrates tothe external surface of the cell as a result of PEF treatment isunimportant. What is important is that should PEFs result in thedischarge of the nucleus from the cell, Annexin-V will stain the PS onthe inside of the plasma membrane, since this stain has access to theinside of the cell due to membrane disruption. Thus, ghost cells shouldhave a bright Annexin-V fluorescence signature. Therefore, gating basedon both PI and Annexin-V has been found by the investigators to give thebest screening for viable cells. Additional markers specific to cancercells can also be included for applications involving cancer cellpurging.

While the above description in association with FIG. 3 is intended toillustrate some representative methods and strategies for performing theinventive cell isolations, it should be understood that the abovedescription is only illustrative and exemplary, and that the inventioncan be performed otherwise than described above without departing fromthe spirit and scope of the invention as presented in the appendedclaims. Also, the above description presents and describes variousmethods and techniques that can for certain embodiments and applicationsbe associated with the invention; however, additional or substitutemethods may be used as apparent to the skilled artisan, and details anddescriptions that, in some cases, may be necessary to perform theinvention but that are known or available to those skilled in the artare not necessarily included or described herein.

The inventive electric field cell/discrete object isolation/inactivationmethods can potentially be performed using a wide variety ofelectroporation equipment known in the art (see for example: Gene PulserII, BioRad, CA; ECM-2001, BTX, CA; Multiporator, Eppendorf, NY;Electroporator II, Invitrogen, CA; PA-4000, Cytopulse, MD). Althoughthere are numerous electroporation systems available, they are notideally suited for cell selection/inactivation discussed herein becausethey have been designed for electroporation or electrofusionapplications for which cell preservation is key, not cell inactivation.They are also typically not capable of processing the number of cellsappropriate for either research or clinical implementations of the cellselection/inactivation strategies discussed herein, since they arelimited by the electric field pulse energies they can deliver. FIG. 4shows one embodiment of a batch system 85 including elements useful forperforming the inventive PEF methods. The main components of theillustrated system are: an electric field pulse driver 101, whichapplies voltage pulses to the PEF electrode enclosure assembly 95 (whichincludes the PEF treatment cell); a power supply 102, which can beexternal or internal to the pulse driver; an optional oscilloscope 103for electric field waveform monitoring; a trigger generator 104; and acontrol/data acquisition system 105, preferably including a computer,for controlling the system and gathering and processing data. Thecontrol/data acquisition system 105, trigger generator 104, oscilloscope103, power supply 102, and pulse generator 101 are electrically coupledvia appropriate electrical connections 106, and together comprise anelectric field generating mechanism 90. The pulse driver 101 of thegenerating mechanism 90 is electrically coupled to the PEF electrodeenclosure assembly 95 via cathode connecting line 100 and anodeconnecting line 99. Also included in the overall system 85 is anoptional forced circulation cooling system 98 which forces cooling fluidthrough the PEF electrode enclosure assembly 95 through lines 96 and 97to remove heat from the treatment cell that is generated by the electricfield and for controlling the temperature of the treatment cell.

The PEF electrode enclosure assembly 95 of system 85 is shown in greaterdetail in FIG. 5. The electrode enclosure assembly 95 includes a PEFtreatment cell 110 that is designed to physically mate with cathode 114and anode 112. FIG. 5 is a cross-sectional view of the PEF electrodeenclosure assembly 95 taken by slicing the PEF electrode enclosureassembly 95, as oriented in FIG. 4, into the plane of the drawing. ThePEF electrode enclosure assembly 95 also includes an annular enclosure122 that is sealingly mated to the top plate 113 and bottom plate 111.Top plate 113 and bottom plate 111 are connected to each other at spacedintervals using a plurality of spacer rods 120 which are transversed bya threaded rod 126 with threadingly attached nuts 121 which may betightened to securely connect the top 113 and bottom 111 plates, orloosened and removed for disassembly of the PEF electrode enclosureassembly 95. Annular enclosure 122 circumscribes and defines an enclosedspace 123 that may be evacuated via evacuation port 125 or pressurizedwith a gas via pressurization port 124 in order to more thoroughlyinsulate the electrical components of the assembly. The treatment cell110 and cathode 114 is supported by a fluid-cooled cathode support rod115 that includes cooling fluid channels 116 and 117. The anode 112 alsopreferably includes fluid cooling channels 118 and 119 to provideadditional heat removal capacity from the treatment cell 110. The fluidcooling channels are in fluid communication with forced circulationcooling system 98 shown in FIG. 4. Forced circulation cooling system 98is preferably sized and designed to maintain the cathode 114 and anode112 at a selected temperature controlled to +/−0.1 degrees C. over atemperature range of at least 4-50 degrees C. Heat removal andtemperature control of the pulsing medium is effected by conductive heattransfer from the medium to the temperature controlled anode 112 andcathode 114. The PEF electrode enclosure assembly 95 should beconstructed with appropriate seals so that enclosed space 123 can bemaintained under high vacuum without significant leakage. The PEFelectrode enclosure assembly 95 can be constructed from a variety ofmaterials apparent to one of skill in the art. The anode assembly 112,and cathode 114, should be constructed from conducting materials such asmetals. In one particular embodiment, the cathode 114 and anode 112 areconstructed of copper. In some preferred embodiments, anode assembly 112is removable from bottom plate 111 to allow easy access and removal oftest cell 110 without the need to disassemble the entire PEF electrodeenclosure assembly 95. The top 113 and bottom 111 plates can beconstructed of any strong stiff material. Preferred plates areconstructed from an insulating material such as a strong plastic, forexample Lexan. Annular enclosure 122 is preferably constructed from atransparent material such as plexiglass.

The static test cell 110 is shown in exploded view in FIG. 6. The testcell comprises an anode end block 131, which mates to anode 112 viaelement 138, a cathode end block 132, which mates to cathode 114 viaelement 144. Each end block includes a plate 137, which is circular inthe illustrated embodiment, and a plurality of holes 136 through whichconnecting elements pass in order to assemble and seal the treatmentcell 110. The end blocks are in contact with plate electrodes 133 and134 which are in turn separated by the fluid containing annular spacer135. Annular spacer 135 includes a channel 139 that, when the treatmentcell 110 is assembled, provides a passage in fluid communication with aninternal volume 140 defined by the annular wall 141 of the spacer 135and the planar walls 142 of plate electrodes 133 and 134. In operation,the cell suspension to be treated is inserted and removed from thetreatment volume 140 via channel 139. The treatment cell 110 is sized toprovide a treatment volume having a desired total volume. Small scaleexperimental systems typically have a treatment volume of about 1-5 ml,while large scale clinical devices preferably have a treatment of 0.1-1liter.

End blocks 131 and 132 can be constructed of any suitable conductingmaterial. Preferred end blocks are constructed from metal. Particularlypreferred end blocks are constructed from copper and subsequently goldplated, or are constructed from tungsten. Annular spacer 135 isconstructed from an insulating material that is preferablybiocompatible. A preferred material for constructing the annular spacer135 is tempered glass (e.g. Pyrex® glass). Electrodes 133 and 134 can beconstructed from any suitable conducting material, with preferredelectrodes being constructed from conducting materials that arebiocompatible and which do not release toxic amounts ofelectro-catalyzed reaction products during application of the electricfield to the sample. Particularly preferred electrodes 133 and 134 areconstructed from graphite carbon. For embodiments utilizing porousgraphite electrodes 133 and 134, the electrodes are preferably degassedafter introduction of a pulsing medium or cell suspension into test cell110 so that bubbles are not released from the electrodes into the mediumduring application of the PEFs. Degassing can be accomplished by avariety of methods apparent to the skilled practitioner, for example thepulsing medium can be added to the assembled test cell 110 atsub-ambient temperature and subsequently heated to ambient orphysiological temperature to release gas bubbles, or the pulsing mediumcan be added to an assembled treatment cell 110 maintained under vacuumduring the adding step. A particularly preferred electrode arrangementthat can reduce or eliminate the release of bubbles trapped in theporous matrix is constructed from graphite which is subsequently sealedwith a sealing agent so that the surfaces 142 in contact with the pulsedsuspensions are rendered essentially non-porous. In preferredembodiments, the porous matrix is sealed with a thin layer of pyrolyticcarbon. The test cell 110 is also preferably constructed and arranged sothat an essentially spatially uniform electric field is applied to thetreatment volume 140. During use, it is important that the suspensionundergoing treatment completely fill treatment volume 140 so that thereis no meniscus that can distort the electric field distribution. Also,preferred treatment cells 110 are constructed and lapped to have verysmooth mating surfaces on components 131, 133, 135, 134, and 132, sothat when assembled, the treatment cell 110 is fluid tight without theneed for supplemental seals, such as washers or O-rings.

A schematic diagram of a preferred continuous flow PEF system 150 isillustrated in FIG. 7. The system includes a flow-through treatment cell151 having two electrodes 152 and 153 in fluid contact with a flowingsuspension being treated that enters the treatment cell 151 through line168 and exits through line 169. The electrodes 152 and 153 areelectrically coupled to a generating mechanism 90, which can beessentially identical in arrangement as that previously described.System 150 also includes pump 160 for pumping cell-free pulsing mediumthrough the system, and pump 161 for pumping a cell suspension throughthe system. Pumps 160 and 161 pump fluid via lines 162 and 163respectively, into line 164, which is in fluid communication withthree-way valve 165. Three-way valve 165 can be set to direct the pumpedfluid to the treatment cell 151 via line 168 or to a waste container 167via line 166. Pulsing medium or treated suspension exiting the treatmentcell 151 via lines 169 and 172 can be controllably directed to a samplecontainer 177 via line 176 or a waste container 175 through adjustmentof three-way valve 173. Particularly preferred embodiments of system 150also include a supplemental pump 170 which controllably pumps a desiredsubstance into the stream of PEF treated cells via line 171. The desiredsubstance can be a variety of materials useful in the fourth step 64through the sixth step 66 of the exemplary procedure 60 discussedpreviously in reference to FIG. 3. For example, the substance suppliedby pump 171 can be DNase or be Trypsin, added to inactivate released DNAand reduce cellular debris, or a medium added to raise the ionicstrength and/or osmolality of the suspension in order to accelerate orbring about inactivation through colloidal osmotic lysis. The system asarranged allows the treatment cell 151 to be initially primed withpulsing medium before addition of cell suspension, and also allows thetreated suspension to be diverted to waste until optimal PEF conditionsare established. Also optionally included in the system 150 but notshown are mechanisms downstream of the treatment cell 151 for removinginactivated cells or cellular debris, such as filters or flowcytometers. In addition, for some embodiments, it may be advantageous toprovide a cooling system, such as system 98 in FIG. 4, to remove heatfrom the flow-through treatment cell 151.

The preferred flow-through treatment cell 151 includes graphiteelectrodes constructed from similar material as discussed for electrodes133 and 134 previously. The electrodes can be enclosed in a flow chamberconstructed of an insulating material such as a plastic, or preferably,Pyrex glass. The electrodes 152 and 153 are elongated in shape and arepositioned so that their long axis is parallel to the direction of fluidflow and have extended planar surfaces 154 and 155 in contact with thefluid in treatment volume 178. The electrodes are preferably constructedand arranged so that the electric field applied to the fluid intreatment volume 178 is substantially spatially uniform and so that theelectric field strength upstream and downstream of the main electricfield treatment region (the region bounded by the portion of theelectrodes 152 and 153 that have surfaces, which are in contact with thecell suspension during operation, that are essentially parallel to oneanother) essentially never exceeds the strength in the main electricfield treatment region. In order to accomplish this, preferredelectrodes include contoured regions 156 and 157 (scale exaggerated inthe Fig.) adjacent the inlet and outlet regions of the test cell 151.Well established electrode profiles exist (e.g. Rogowski, Ernst, orChang profiles) that can be implemented to obtain the purpose ofcontours 156 and 157. The treatment volume 178 of the test cell 151 isdefined, for rectangularly shaped test cells, as the product of thelength 179, the gap width 195, and the height h (into the plane of thefigure and not shown) so that v_(TV)≈lwh, where v_(TV) is the totalvolumetric capacity of treatment volume 178, l is the length of the testcell 151, and w is smallest gap width 195 between electrodes 152 and153. These dimensions are chosen for a particular application to yield adesired volumetric throughput having a desired total treatment residencetime in the treatment volume 178 and a shear rate below the value thatcan cause damage to the cells. For a test cell having an essentiallyuniform rectangular cross-section for flow, the average residence timeτ_(res) of fluid in the treatment volume v_(TV) is:

τ_(res) =v _(TV) /u=lwh/u  (17)

where u is the volumetric flow rate of the fluid. The maximum laminarshear rate Γ can be approximated by:

Γ=2u(w+h)/(wh)²  (18)

The treatment cell dimensions and throughput flow rate should beselected to provide a desired total exposure time t to the electricfield for a given applied pulse duration, τ_(p), and pulse frequency F.For example, if the desired total treatment time is t, then thenecessary pulse frequency would be:

F=(ut)/(lwhτ _(p))  (19)

For one exemplary embodiment of treatment 151, length 179 is 37.3 mm,the gap width 195 is 8 mm, and the height (into the plane of the figure)is 4 mm.

Startup and operation of the flow-through system 150 can proceed asfollows. The system, in this exemplary embodiment, employs syringe pumpsfor pumps 160, 161, and 170. With pump 160 loaded with cell-free pulsingmedium, pump 161 loaded with the cell suspension to be treated, and pump170 loaded with pulsing medium supplemented with DNase, three-way valve165 is positioned to direct fluid to the treatment cell 151, andthree-way valve 173 is positioned to direct fluid to waste container175, and the lines and treatment cell 151 is flooded with pulsing bufferby activating pump 160 in order to remove bubbles from the system. Next,pump 160 is stopped, three-way valve 165 is repositioned to direct fluidto waste container 167, and pump 161 is activated to pump cellsuspension to waste container 167 in order to remove any bubbles fromline 163. Three-way valve 165 is then switched to direct the cellsuspension to the treatment cell 151 and the generating mechanism 90 isactivated to apply PEFs to the treatment volume 178. As soon as it isdetermined that the system is functioning properly, three-way valve 173is positioned to direct the treated cell suspension into cell collectioncontainer 177. When a desired quantity of cell suspension has beenprocessed, pump 161 is shut off and the generating mechanism 90 isswitched off. Pump 170 may be operated as desired during PEF treatmentto add DNase to the treated cell suspension to prevent coagulation ofany cellular debris. The sequence of events just described is preferablycomputer controlled and pertinent PEF system data is automaticallycollected and stored by a data acquisition system. In alternateembodiments of flow through system 150, instead of passing through thetreatment cell 151 only once, the cell suspension can be recycled backto the inlet of the treatment cell for a plurality of PEF treatments.Also in some embodiments, instead of supplying a pulsed electric fieldto the treatment volume 178, the field is maintained at an essentiallyconstant value during the PEF subjecting step, and the average exposuretime of the cells in the suspension is simply the average residence timeof the cells in the treatment volume 178 determined by the suspensionflow rate.

Pulse driver 101 (see FIG. 4) for use in systems 85 and 150 can be anysuitable pulse driver known in the art that is capable of producingpulsed electric fields within the PEF treatment volume of sufficientmagnitude and duration for inactivating discrete objects of interest.Preferred pulse generators are able to induce an electric fieldmagnitude of at least about 5 kV/cm, more preferably at least about 10kV/cm, and most preferably at least 20 kV/cm in the treatment volume.Preferred pulse generators are able to supply an electric field pulseduration above a set point electric field strength of between about 2-20μs at pulse frequencies between about 0 to 10 kHz. Preferred pulsedrivers are able to produce a substantially rectangular pulse shape, aspreviously discussed, with rise and fall times not exceeding about 0.5μs. Preferred pulse driver mechanisms also include control circuitry toallow the maximum pulse voltage to be controllable by the user and toterminate pulsing upon detection of an electrical short circuit or arc.While pulse drivers based on gas switches, such as thyratrons and sparkgaps, can be employed for use in the invention, because of theirtypically superior durability and reliability, pulse drivers based onall-solid-state switch technology are preferred.

Design specifications for the pulse driver are determined from therequired maximum applied electric field strength, maximum requiredelectric field pulse duration, the size of the treatment volume, and theelectrical resistivity of the suspensions being treated. The pulsedriver for use in a particular application must be designed to supply amaximum pulse energy, e_(p)(Joules/pulse), determined by:

e _(p)=τ_(p) v _(TV) E ₂/ρ_(ps)  (20)

where τ_(p) is the maximum pulse duration, v_(TV) is the treatmentvolume, E is the maximum required electric field strength, and ρ_(ps) isthe resistivity of the sample being treated. For impedance matchedconditions, the load voltage, V_(l), is one-half the maximum chargevoltage of the pulse driver. The load voltage requirements of the pulsedriver can be determined by:

V _(l) =Ew  (21)

where E is the maximum required electric field strength, and w is thedistance between the electrodes (see FIG. 7). The resistance load of thetreatment cell which the pulse driver must be able to handle isdetermined by:

R=ρ _(ps) w ² /v _(TV)  (22)

where R is the impedance load (Ω), ρ_(ps) is the resistivity of thesample being treated, w is the distance between the electrodes, andv_(TV) is the treatment region volume.

FIG. 8 is a block diagram of one embodiment of a pulse driver system 180for use in the inventive PEF systems. The pulse driver 180 produces thesubstantially rectangular bipolar pulse shown in FIG. 9. The bipolarrectangular pulse shown in FIG. 9 has short rise 201 and fall 202 timesseparated by flat regions 203 of substantially constant voltage. Thetrailing opposite polarity segment 204 of the waveform serves to reduceand/or reverse electrochemical reactions that otherwise can producehydrogen and chlorine bubbles, which can degrade performance and affectsuspension pH. In other embodiments, instead of supplying a bipolarpulse as shown in FIG. 9, the pulse generator may instead supply aunipolar pulse, as shown in FIG. 10, while simultaneously supplying areverse polarity DC current properly matched to essentially equal thetime-averaged current of the electrical field pulses. The pulse shapeshown in FIG. 10 is not substantially rectangular in shape but isinstead in the shape of a half sine-wave. As previously discussed, suchpulse shapes will, in general, yield poorer electroporation thresholdselectivity than more rectangularly shaped pulses.

The pulse driver system in FIG. 8 that produces the waveform shape shownin FIG. 9 includes two pulse driver circuits 205 and 206, one 205 for apositive polarity voltage pulse, the other 206 for a trailing, negativepolarity pulse. Each of the pulse drivers includes a DC power supply(182 and 183), a storage capacitor 184, and a stack of integratedbipolar transistors (IGBTs) 185 and 187, which are solid state switchesthat apply the electrical energy stored in the storage capacitors 184 tothe PEF treatment cell 191. The IGBTs each include a switch stack andgate trigger 186, 188, and a diode 189, 190 on the output line. TheIGBTs are stacked in series and parallel combinations to provideenhanced voltage and current capabilities. Power is supplied to thepulse driver system from a source 181 of facility line power. Preferredpulse driver systems also include circuitry 193 for process control andcircuitry 192 for system diagnostics and data acquisition as known inthe art. In preferred embodiments, the electric field strength developedin the PEF treatment cell 191 is determined by circuitry 192 for systemdiagnostics and data acquisition by use of a calibrated high voltageprobe/oscilloscope system. The current wareform can be measured using aPearson coil and is, in preferred embodiments, recorded on the sameoscilloscope as the voltage waveform. The time integrated product of thecurrent and voltage waveforms can then be used to determine pulseenergy. Pulse energy thus determined, together with measurements of thepulse repetition frequency and the temperature of the electrodes in thetreatment cell 191 can be used, together with a heat transfermathematical model describing the heat transfer characteristics of thetreatment cell 191, to calculate the temperature evolution of the pulsedsuspension. Alternatively, the temperature of the pulsed suspension canbe directly measured in a plurality of locations within the treatmentvolume with suitable temperature probes or thermocouples. Diagnostic 192and control 193 systems also, in preferred embodiments, include one ormore computer elements, for example integrated personal computers, tocontrol system operation and perform data reduction and analysis.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples below. Thefollowing examples are intended to illustrate the operation of thepresent invention, but not to exemplify the full scope of the invention.

EXAMPLES Introduction

The experimental examples that follow, presented as five separate cases,demonstrate pulsed electric field cell-size selectivity, hematopoieticprimitive progenitor cell enrichment, and tumor cell purging. Two PEFcell-size selection examples are presented (Cases 1 and 2). In Case 1PEFs were applied to a suspension of peripheral blood mononuclear cellsresulting in a step-wise reduction in size distribution of the PEFtreated viable cells as a function of electric field strength and totalelectric field exposure time. In Case 2 PEFs were applied to asuspension of peripheral blood mononuclear cells, illustratinglymphocyte enrichment by selective inactivation of monocytes in PEFtreated specimens as a function of electric field strength and electricfield exposure time. Case 3 illustrates the enrichment of hematopoicticstem cells in PEF treated peripheral blood progenitor cell specimens. InCase 3, PEFs were applied to mobilized peripheral blood specimensharvested from a patient by leukopheresis. Case 3 illustrates thedependence of stem cell enrichment on electric field strength for afixed electric field exposure time. Two cases are presented thatillustrate PEF tumor cell purging (Cases 4 and 5). In Case 4, PEFs wereapplied to a suspension of peripheral blood mononuclear cells seededwith a megakaryocyte tumor cell line (CMK). This case illustrates theselective inactivation of the tumor cells with simultaneous preservationof lymphocyte and monocyte cells as a function of electric fieldstrength and fixed total electric field exposure time. In Case 5 PEFswere applied to a suspension containing only mammary gland breast tumorcells (MCF-7). This case illustrates the PEF inactivationcharacteristics of this breast tumor line as a function of electricfield strength, pulse duration, and total electric field exposure time.The examples given by Cases 1-5 were performed under non-optimized PEFconditions. Thus, these results represent a lower end demonstration ofthe capability of the PEF cell selection strategy.

Methods and Materials

The methods and materials for the five example cases are given togetherbelow and are broken into three categories: cell preparations, cellassays, and PEF apparatus.

Cell Preparations

Four cellular systems were used in the example cases: peripheral bloodmononuclear cells (PBMCs), mobilized peripheral blood mononuclear cells(hereafter referred to as peripheral blood progenitor cells, (PBPCs)),megakaryocyte tumor cells (CMKs), and mammary gland breast tumor cells(MCF-7s).

PBMCs were used in Cases 1, 2, and 4. The PBMCs were obtained fromhealthy donors by harvesting approximately 60 ml of peripheral blood pertest day by venipuncture through a 21 G, 1 inch needle (BecktonDickinson, 305175) into a syringe (Beckton Dickinson, 309663) containing1 ml Heparin (5000U, Elkins-Sinn, Inc., A-0400H). The PBMCs cells wereseparated from the whole blood using standard density gradientcentrifugation techniques. Under sterile conditions, the blood wasdiluted with twice its volume with 1×phosphate buffered saline (PBS,Mediatech, 21-031-CV) and then aliquoted (30-40 ml) into 50 ml conicalcentrifuge tubes. Ficoll-Paque (Pharmacia Biotech, 17-0840-03) wasslowly dispensed into the bottom of each centrifuge tube, and the tubeswere centrifuged (IEC, Centra GP8R, rotor no. 228) at 2000 RPM for 30minutes with the brake off. The PBMCs at the density interface werecollected by aspiration with a 5 ml pipet. The recovered PBMC layerswere combined into a single 50 ml centrifuge tube. The resulting PBMCsuspension was then diluted 10× using PBS, aliquotting into as many 50ml centrifuge tubes as required, followed by centrifugation at 1500 RPMfor 5 minutes. The supernatant was aspirated, and one pellet wasresuspended in 10 ml PBS. This 10 ml suspension was then used toresuspend the pellets of all remaining tubes. PBS was then added to theresulting suspension, bringing the total volume to 50 ml. This 50 mlsuspension was centifuged at 1500 RPM for 5 minutes; the supernatant wasaspirated; the pellet was then resuspended in 20 ml IMDM (Sigma, I 2762)with 10% fetal calf serum (FCS, Sigma, F 2442), and then transferred toa flask and placed in an incubator at 37° C. overnight in preparationfor PEF treatment the next day. The next morning the contents of theflask were transferred to a 50 ml centrifuge tube. The flask was washedthree times with 10 ml PBS: these PBS wash volumes were added to the 50ml tube containing the bulk of the PBMC specimen. The resultingsuspension was then centrifuged at 1500 RPM for 5 minutes; thesupernatant was aspirated, and the cells were suspended in the desiredpulsing buffer. Pulsing and post-PEF treatment buffers are discussed inmore detail in a subsequent section.

Cell preparation for Case 3 was identical to that for Cases 1 and 2 withthe following exceptions. The cells for Case 3 were obtained byleukopheresis from a cancer patient that had been administeredgranulocyte colony stimulating factor (G-CSF). More specifically,mobilization of the patient's peripheral blood was effected byadministering G-CSF (10 μg per kg body weight per day) subcutaneouslyfor 4-6 days, with apheresis collections beginning on day 4 until2.5×10⁶ CD34+ cells per kg body weight were obtained. The resulting cellsuspension contained approximately 2×10⁸ leukocytes in approximately 2ml PBS. This cell preparation was used, instead of the whole blood inthe protocol described above for obtaining PBMC preparations. Beforesuspending the PBPCs in the pulsing buffer, the resting lymphocytepopulation was activated to move these cells to their larger, activestate in order to improve for stem cell enrichment. To activate thelymphocytes, the PBPC suspension, interleukin-2 (IL-2, 50 IU/ml, R n′ DSystems) and PHA (0.25 μg/ml, Sigma) were added to a culture medium (20ml, IMDM, 10% FCS) and incubated at 37° C. for 36 hours. Afteractivation (incubation), the suspension was centrifuged at 1500 RPM for5 minutes, the supernatant was aspirated, and the resulting pellet wasresuspended in the desired pulsing buffer.

For Case 4, PBMCs, prepared as described above for Cases 1,2 and 4, wereseeded with a megakaryocyte tumor cell line (CMK, Sato T. et al., Br. J.Haematol. June; 1989 72(2): 184-90). For Case 5, a suspension containingonly mammary gland tumor cells (MCF-7, ATCC no. HTB-22) was exposed toPEFs. Preparation of the MCF-7s for PEF treatment is described belowwith modifications to the procedure for the CMK line noted. MCF-7 (ATCCno. HTB-22) cells were thawed under the following conditions. A vial ofcells was thawed at 37° C., then transferred to a 50 ml tube (Fisher,14-959-49A) containing 30 ml of 1×Iscove's medium without glutamine(IMDM, Fisher, MT15 016L,V) and centrifuged at 1500 RPM for five minutesat room temperature. The supernatant was discarded and the pellet wasresuspended in 20 ml of MCF-7 culture-medium and cultured at 37° C., 5%CO₂. The MCF-7 culture medium used was 1×Dulbecco's modification ofEagle's medium, without glutamine, including 4.5 g/L glucose, andsupplemented with 2 mM L-glutamine (Fisher/Cellgro. MT-25-005-LI), 50I.U./ml of penicillin, and 50 μg/ml streptomycin (Fisher/Cellgro,MT-300-01-LI) and 20% FCS. The cells were split upon reachingconfluence, typically every three to four days, as follows. Culturesupernatant was aspirated and replaced with 5-6 ml of trypsin (2.5 g/L1:250 in HBSS without calcium or magnesium, Fisher/Cellgro, 25-050-11).After incubation at room temperature for 5 minutes, the flask was rinsedusing 10 ml of 1×Dulbecco's Phosphate buffered saline without calcium ormagnesium (PBS, Fisher/Cellgro, MT-21-031-CV) supplemented with 1% fetalcalf serum (FCS, Sigma, T-2442). The rinse solution was then transferredto a 50 ml tube, and the volume was made up to 50 ml with PBS,supplemented with 1% FCS, before centrifuging the tube, as describedabove. After centrifugation, the supernatant was discarded and thepellet was resuspended in 10 ml of MCF-7 culture medium. Fivemilliliters of this suspension was transferred to one of two 75 cm²flasks, each containing 15 ml of MCF-7 culture-medium, prior toreculture under the above described conditions. If there were more thantwo flasks to be split, then the excess cells were frozen as follows.The cells were trypsinized and washed as previously described, and thenresuspended in 1 ml of freezing medium (10% DMSO, Fisher, D128-500, 90%FCS), per flask and transferred to labeled cryovials (Fisher, 5000-1020)prior to transfer to a −70° C. freezer. MCF-7 PEF treatment commencedthe morning after these cells achieved half-confluency in the cultureflasks. On the morning of a PEF experiment, the MCF-7s were trypsinizedusing the above protocol, the recovered cells were then washed usingPBS, and the centrifuge pellet was then suspended in pulsing buffer inpreparation for PEF treatment.

For the CMK line, the cell suspensions were thawed as previouslydescribed for the MCF-7 line and cultured in CMK culture medium at 37°C., 5% CO₂. The CMK culture medium comprised 1×RPMI 1640 medium, withoutglutamine, supplemented with 2 mM L-glutamine (Fisher/Cellgro,MT-25-005-LI), 50 I.U./ml of penicillin, and 50 μg/ml streptomycin(Fisher/Cellgro, MT-300-01-LI) and 20% FCS. The CMKs were split 1:10every 3-4 days by transferring about 2 ml of the cell suspension to anew 75 cm² flask containing 18 ml of CMK culture medium. Excess cellswere frozen as previously described. On the morning of a PEF treatmentexperiment, the CMKs were transferred from the culture flasks to 50 mlFalcon tubes (Becton Dickinson, 2098). As part of this transfer, thecells were passed through a separation filter (Miltenyi Biotec, Inc.,414-07) to remove large-scale debris. The cells were then washed in PBSand resuspended in the pulsing buffer in preparation for PEF treatment.

Pulsing Medium

For Cases 1 and 2, IMDM was used as the medium in which the PBMCs weresuspended for PEF treatment. After PEF treatment, the treated specimenswere combined with an equal volume of IMDM. The resulting specimenremained at room temperature until preparation for flow cytometryanalysis.

For Cases 3, 4, and 5, the cells were suspended in a low ionic strengthmedium (10% v/v PBS, 90% v/v isotonic sucrose solution). This low ionicstrength pulsing medium was formulated to be isotonic. After PEFtreatment, the treated specimen was combined with an approximately equalvolume of IMDM. The inactivation protocol using a low ionic strengthpulsing medium, followed by resuspension in a higher ionic strengthmedium, was used in these cases to investigate whether the combinationwould result in more extensive post-PEF fragmentation of PEF poratedcells by colloidal osmotic lysis. The low ionic strength pulsing bufferwas formulated as follows. Twenty milliliters of 1×sterile PBS wascombined with 180 ml of distilled/deionized water. To this solution,16.6 g of powdered sucrose was added (Fisher, BP220-212). This solutionwas then sterilized by passing it through a 0.2 μm filter (Nalgene,291-3320). The pH of the resulting solution was checked using a BeckmanΦ40 pH meter and was found typically to lie in the range 7.4-7.6. Priorto final preparation of the cellular suspensions for pulsing, trypanblue exclusion using phase contrast microscopy was employed to enumeratethe number of viable cells present in PBS suspensions of the variouscells. Given the number of cells desired in the cellular suspensions forpulsing, the trypan blue results were used to determine the volume ofPBS cell suspension required to provide the desired number of cells,which volume was then centrifuged to pellet the cells. The pellet wasthen suspended in the required amount of pulsing buffer.

Cell Assays

Pre- and post-PEF cell enumerations were performed using both trypanblue exclusion, using a hemacytometer (Fisher, 02-671-10) under phasecontrast optical microscopy, and a wide variety of well established flowcytometry protocols. Trypan blue exclusion was used primarily fordetermining the number of viable cells to suspend in the pulsing mediumfor each experiment. Flow cytometry, using a variety of viability andconjugate antibody fluorescent stains, was used to enumerate pre- andpost-PEF viable cell numbers, including lymphocytes (B- and T-cells),monocytes, primitive progenitor cells (including stem cells), and tumorcells (CMK and MCF-7). A Becton Dickinson FACScan flow cytometer wasused to perform the analytical assays. For Cases 1, 2, 3, and 4, cellviability was determined by flow cytometry using either propidium iodide(PI, Molecular Probes, P-3566) or TO-PRO-3 (Molecular Probes, T-3605)DNA staining combined with light scatter characteristics. Viablelymphocytes were identified as those cells scoring low for theparticular DNA viability stain used (either PI or TO-PRO-3) whilestaining brightly for CD3 (T-cells, Fisher, OB9515-02 or -09) or CD19(B-cells, Fisher, Becton Dickinson, 340409 or 340364). Viable monocyteswere identified as those cells scoring low for the DNA viability stainused while staining brightly for either CD11b (Becton Dickinson,347557), CD13 (Fisher, OB9555-02 or -09), or CD14 (Fisher, OB9560-02 or-09) and CD45(leukocytes, Fisher, OB9625-02 or -09). Viablehematopoietic stem cells were identified as those cells scoring low forthe DNA viability stain used while staining brightly for CD34)Fisher,OB9595-02 or -09and dimly for CD38)Fisher, OB9610-02 or -09. Viable CMKtumor cells were identified as those cells scoring low for the DNAviability stain used, staining dimly for CD14, and staining brightly forCD45, while also being outside of the light scatter compartments forlymphocytes and monocytes (light scatter gating).

For Case 5, involving the PEF inactivation characteristics of MCF-7tumor cells, viable MCF-7s were identified as those cells scoring lowfor both the apoptotic membrane stain Annexin-V (Caltag, Annexin VV01-3)and the DNA stain PI. Light scatter gating was also used to discriminatecells from cell debris that was smaller than the smallest of the MCF-7cells. Both PI and Annexin-V viability stains were used for MCF-7analysis based on the following considerations. Propidium iodide is aDNA stain. Thus, for cells with disrupted membranes, yet stillcontaining a nucleus, this dye will stain non-viable cells Annexin-V,however, stains phosphatidylserine (PS) which migrates from the insideto the outside of the plasma membrane during normal apoptosis. Thus, itis normally used as an indicator of apoptotic, but still viable cells.Whether or not the PS migrates to the external surface of the cell as aresult of PEF treatment is unimportant. What is important is that shouldPEFs result in the discharge of the nucleus from the cell, Annexin-Vwill stain the PS on the inside of the plasma membrane, since this stainhas access to the inside of the cell due to membrane disruption. Thus,ghost cells should have a bright Annexin-V fluorescence signature.Therefore, gating based on both PI and Annexin-V has been found by theinventors to give the best screening for viable MCF-7 cells.

Test and Control Specimens

On a given test day, both control specimens and PEF treated specimenswere prepared. Two types of controls were prepared: 1) a stock cellcontrol specimen and 2) a PEF test cell control specimen. Stock cellsuspension refers to the cellular suspension in pulsing medium fromwhich fixed volume aliquots were taken for loading into the PEFtreatment cells. The stock cell suspension controls were prepared byplacing the same volume of stock cell suspension into a 15 ml centrifugetube as would be loaded into the PEF treatment volume. An additional 5ml of IMDM was then added to the same tube. The PEF treatment cellcontrols were prepared by loading a PEF treatment cell with theappropriate volume of cell suspension and allowing it to stand at roomtemperature for the period of time it would normally take to treat aspecimen with PEFs; however, during this time, no PEFs were applied tothe PEF treatment cell control specimens. When the standing periodexpired, the specimen was removed from the PEF treatment cell and placedin a 15 ml centrifuge tube to which 5 ml of IMDM was added. The PEFtreated specimens were handled in the same way as the PEE treatment cellcontrols, except PEFs were applied to the treated specimens.

The control specimens served two purposes. First, a comparison of thetotal viable cell counts, as provided by flow cytometry, between thestock cell controls and treatment cell controls gave an indication ofthe fraction of cells lost simply by virtue of residence in the PEFtreatment cells. Second, the cytometry counts from the treatment cellcontrols for each cell type were used to normalize the cytometry countsfor each cell type for the PEF treated specimens. This allowed forcomputation of the surviving percent for each cell type for each set ofPEF conditions. To obtain consistent and meaningful cytometry counts,the following general procedure was followed. By virtue of the fact thatthe PEF treatment cells employed had a fixed volume, and that the stockcell suspension controls were prepared using a stock cell specimenhaving the same volume as the PEF treatment cells, all of the specimensprepared on a given test day had the same number of input cells. Whenpreparing the control and PEF treated specimens for flow cytometryanalysis, all cellular material present prior to PEF treatment wasultimately contained in the aliquot the cytometer drew from whenperforming the flow cytometry assays. Thus, if the volume of eachcytometry aliquot was the same for all specimens on a given test day,then acquiring counts for a fixed acquisition time for all specimenswould provide viable counts that could be compared across the specimensprepared on a given test day. Most importantly, the viable counts forthe treatment cell controls could then be used to normalize the viablecounts for each PEF treated specimen, thereby providing the informationneeded to compute surviving percent for each PEF-exposed specimen.

Flow Cytometry

The following procedures were used during preparation of the control andPEF-treated specimens for flow cytometry analysis. 100 μl of DNase I(100 mg; 500 Kunitz-units per mg solid, Sigma, DN-25) was added to eachspecimen to minimize coagulation by released DNA. The specimens werethen vortexed and centrifuged for 6 minutes at 1500 RPM. The supernatantwas aspirated and the pellet was washed with 5 ml PBS. When preparingfor PI and Annexin-V staining, the specimens were centrifuged again for6 minutes at 1500 RPM; the supernatant was aspirated, and 100 μl of1×calcium buffer was added (Bender MedSystems, BMS306BB), followed byaddition of 150 μl of propidium iodide (PE conjugate) and 5 μl ofAnnexin-V (FITC conjugate). These specimens were incubated at roomtemperature for 15 minutes. Next, 300 μl of 1×calcium buffer was added,the sample vortexed, and then the specimens analyzed under flowcytometry. PI/Annexin-V staining was the only staining used for Case 5where the PEF inactivation characteristics of MCF-7s (with no othercellular species resent) is presented. PI staining, in conjunction withother conjugate monoclonal antibody stain, was used for Cases 1 and 2,where the inactivation of PBMCs is presented: Annexin-V viabilitystaining was not used for these cases (Cases 1 and 2). For Case 1 thespecimens were also stained using the conjugate monoclonal antibodiesCD3 and CD11b in order to enumerate lymphocyte (T-cells) andmonocyte/granulocyte cells, respectively. For Case 2, the specimens werealso stained with the conjugate monoclonal antibodies CD3 and CD13 inorder to enumerate lymphocyte (T-cells) and monocyte cells,respectively. For Case 3, the washed pellets were resuspended in 200 μlPBS, which was then aliquoted to two 100 μl specimens. The conjugatemonoclonal antibody stains CD14 and CD45 were added to one of thesealiquots for enumeration of lymphocyte and monocyte cells, respectively.The conjugate monoclonal antibody stains CD34 and CD38 were added to thesecond aliquot for enumeration of primitive progenitor cells(hematopoietic stem cells). Both aliquots were then incubated at roomtemperature for 15 minutes. These aliquots were then washed with 2 mlPBS, centrifuged for 6 minutes at 1500 RPM; the supernatant was thenaspirated, and 500 μl of 1 μg/ml TO-PRO-3 viability stain was added tothe tube. The resulting aliquots were then incubated for 15 minutes atroom temperature, vortexed, and analyzed under flow cytometry. Thestaining for Case 4 was very similar to Case 3, except the specimenswere not stained with the CD34/CD38 conjugate monoclonal antibodies.Rather, these specimens were stained with the conjugate monoclonalantibodies CD3 and CD19 (in addition to CD14 and CD45) in order toenumerate T- and B-cells, respectively.

When analyzing Cases 1, 2, 4, and 5 under flow cytometry, approximately100,000 cytometer events were found to give adequate resolution of thecell types of interest (lymphocytes, monocytes, tumor cells). Due totheir rare frequency, however, 500,000-1,000,000 cytometer counts wererequired to identify the CD34+/CD38− stem cell population for Case 3.

PEF Apparatus

The apparatus used for the examples that follow was a batch PEFtreatment system, i.e., PEF treatment takes place in a fixed, static PEFtreatment volume. The batch PEF system is comprised of a batch PEFtreatment cell, an electrode enclosure assembly that holds the PEFtreatment volume during exposure to PEFs, an electric field pulsegenerator that applies voltage pulses to the PEF treatment cell, and acomputer control/data acquisition system.

The essential details of the PEF treatment cell used for the examples isillustrated in FIG. 6. The treatment volume is formed by stacking theend blocks 131, 132, graphite disk electrodes 133, 134, and Pyrexannular spacer 135 as shown in FIG. 6. This treatment cell has asealless design, i.e., no O-ring or gasket seals are employed to sealthe PEF treatment volume 140, defined by the mating of the Pyrex annularspacer and graphite disks. Rather, all mating surfaces were lapped to awaviness of no more than 2.5 μm over the diameter (8.26 cm) of the disksinvolved the assembly. Furthermore, all mating surfaces were lapped to anumber 4 surface finish, which had an RMS variation in surfacedeviations of +/−0.1 μm. The graphite disks were made of ISO-63graphite, which had an average pore size of 1 μm. The tolerances werechosen to prevent blood cells, which are typically greater than 5 μm,from finding their way into the interface regions of the matingtreatment cell components, or penetrating into the pores of the graphitedisk electrodes. Nylon screws were employed to hold the treatment cellassembly together. Since all internal surfaces of the treatment cellwere either normal to the electric field direction (e.g. conductingsurfaces) or were parallel to the electric field direction (e.g.insulating surfaces), the utilized treatment cell geometry provided veryuniform electric field strength over the entire treatment volume. Theend blocks of the treatment cell were made of gold coated copper. Thegold coating was included to minimize surface oxidation and to provideexcellent electrical contact with the assembly that holds the treatmentcells during PEF treatment. The pulsing suspension was loaded andunloaded from the PEF treatment cell through the radial channel 139 inthe Pyrex annular spacer by using a pipetting syringe (pipettingneedles, Fisher, 14-825-16N); 10 ml disposable syringes, Fisher,14-823-2A).

The treatment cell just described was used for Cases 1, 2, 3, and 4. Itis referred to hereafter as the Type A test cell. For Case 5, a variantof the Type A test cell was used. This variant is referred to as theType B test cell (not shown) and was similar to the Type A test cellpreviously described with the following differences. The type B testcell used tungsten electrodes, rather than graphite and the annularspacer was made of plexiglass that had four radial holes (1 mm I.D.),equally spaced around its perimeter, that penetrated from the outersurface through to the inner surface. A silicon washer, which had anoutside diameter equal to the inside diameter of the plexiglass spacerand which was approximately 0.05 mm thicker than the plexiglass annularspacer, was inserted inside the plexiglass spacer during assembly of theType B test cell, which silicon washer ultimately formed thecircumferential bounding wall of the PEF treatment volume. Once the TypeB test cells were assembled, they were loaded and unloaded with cellsuspension through the 1 mm I.d. holes using A 21 G hypodermic needlesmated to 1 ml syringes (21 G needles, Becton Dickinson, 305176; 1 mlsyringes, Becton Dickinson, 5602).

The internal dimensions of the test cells that were critical for theheat transfer and electric field strength calculations, were as follows.For the Type A test cells, the diameter of the cylindrical treatmentvolume was 4.45 cm, and the gap between graphite electrodes, when thetest cell is assembled, was 3.2 mm, which yields a volume of 4.94 ml.For the Type B test cells, the diameter of the cylindrical treatmentvolume was 1.91 cm, and the gap between graphite electrode was 2.5 mm,which yields a volume of 0.72 ml. The parts making up the test cellswere fabricated using standard methods known in the art.

In general, electrochemical reactions take place in the region near theelectrode surfaces when a current is passed through the electrodes intoan electrolyte solution. For the present application, were thousands ofelectric field pulses may be applied, gold is not a good electrodecandidate. This is because free chlorine ions can react with the gold,forming a gold salt, which dissolves in the electrolyte. Tungsten is areasonably inert electrode material, but it can react with free oxygenions to form an oxide layer on the electrodes. Carbon electrodes(graphite) are the preferred electrode material since graphite issubstantially inert with respect to the ionic species formed during PEFtreatment.

To demonstrate to the importance of controlling electrochemicalreactions, separate experiments were performed where electric fieldpulses were applied to isotonic potassium chloride solutions. Plumes ofbubbles were observed to be released from both the anode and cathodeelectrodes of the Type A test cell. Furthermore, after a thousandpulses, the pH of the solution shifted from 7.0 to 6.0. However, when aDC counter-current was driven through the test cell during pulsing,which had a current equal to and opposite of the time average of theprimary electric field pulses, no bubble formation was detected and theswing in pH was undetectable. These experiments illustrated the abilityof using a reverse polarity current to control electrochemical reactionsin unbuffered aqueous solutions having high (i.e. physiological) ionicstrengths. Reverse polarity currents were not employed in the examplecases to follow since the pulsing media used for those cases wastypically of lower ionic strength and included a buffer to counter theeffects of low concentrations of electrochemically produced products.

Once the test cells were loaded with the desired cellular suspension,the test cells were then mounted in an electrode/enclosure assembly thatsupports the test cell when the PEFs are applied to the test cell. Theelectrode/enclosure assembly used for the example cases following hasessentially the same features as the assembly shown in FIG. 5. Thisassembly serves three purposes. First, it provides electrical contactbetween the electric pulse generator and the PEF treatment cell. Second,it provides temperature control for the test cell. Third, it enclosesthe test cell during PEF treatment, thereby protecting personnel inclose proximity to the system from high voltage components. Withreference to FIG. 5, the temperature of the test cells were controlledby contact with temperature-controlled cathode 114 and anode 111supports of the electrode/enclosure assembly 95. The temperature of thecathode and anode supports is controlled by circulating temperaturecontrolled water through these structures using a heater/chiller system(Neslab, RTE-110). This system is capable of setting the temperature ofthe test cells to +/−0.2° C. over the range 0-50° C. After mounting thetest cells in the electrode/enclosure assembly, the test cells wereallowed to reach thermal equilibrium with the temperature of the cathodeand anode structures in the electrode/enclosure assembly.

The electric pulse generator system used for the examples consisted ofan energy storage capacitor (3 kV, 500 μf, Maxwell, 38683), which wasconnected to an all-solid-state pulse driver designed and fabricated bythe inventors for driving a CO₂ laser. The pulse driver (electric pulsegenerator) is referred to as the COLD-I. This driver switches the energystored in the 500 μf capacitor via solid-state SCR switches to a 20:1saturable core transformer into an impedance matched load (˜8Ω). Thisdriver, as presently configured, can deliver fixed duration voltagepulses (˜5 μs duration) at voltages up to 8 kV. Under these conditions,the deliverable pulse energy is ˜80 J. The storage capacitor was chargedvia a Maxwell (1 kV, 5 kJ/s, CCDS 501P372-208) high voltage chargingpower supply. Normally, the voltage output of the COLD-I is to high foruse for PEF cell selection. This was remedied by using a high powervoltage divider circuit, which simply comprised two resistors in seriesconnected to ground. This voltage divider circuit could attenuate thevoltage pulses delivered to the PEF test cell by factors of 4-20 simplyby changing the resistance of the two resisters in the circuit.

A Pearson coil current transducer was used to monitor the currentdelivered to the PEF test cell. A second Pearson coil transducermeasured the current through a precision 100Ω resister connected acrossthe PEF test cell. The voltage applied to the test cell was derived fromthe second Pearson coil transducer signal by multiplying it by 100Ω.These current and voltage signals were displayed on a LeCroyoscilloscope (9410). FIG. 10 presents the voltage waveform produced bythe COLD-I driver when applied to a Type A test cell containing a roomtemperature isotonic potassium chloride solution, which corresponds toimposing a 2.2 kV/cm electric field in this saline solution.

An IBM personal computer clone was used for system control and dataacquisition. During PEF treatment, a control program was run that issuedpulse driver trigger signals. The frequency and number of the triggersignals was set by the operator based on the PEF conditions desired. Themagnitude of the voltage pulses delivered to the PEF test cell, however,was set by a dial associated with the Maxwell charging power supply. Theoutput trigger signal from the PC control computer was delivered to anopto-isolator circuit, which prevented any electric noise from feedingback to the PC. The output of the opto-isolator circuit was delivered toan HP 214-B trigger generator, which, in turn, sent a 100 volt, 2 μspulse to the COLD-I driver, which triggered the COLD-I, thereby applyingthe desired electric field pulse to the PEF test cell.

Upon completion of a PEF treatment experimental trial, the voltage andcurrent waveform data was downloaded from the oscilloscope to the PCcomputer via a GPIB interface. After the data was downloaded,post-processing computations were performed. More specifically, thefollowing quantities were computed: peak voltages and currents, theresistance of the test cell, the full-width at half-maximum (FWHM)electric field pulse length, and the energy delivered to the test cellper electric field pulse. For the cases that follow, the electric fieldstrengths quoted were those based on the peak voltage of the pulsesdelivered to the PEF test cell. Furthermore, the electric field pulselengths quoted were the FWHM values. After computing the single pulseenergy deposited to the test cell, the average midplane temperature risein the PEF test cell and the temperature jump per electric field pulsefor a given PEF treatment trial was computed. These temperatures, whenquoted in the cases that follow, were derived using Eqs. 14, 14a, and14b given earlier in the detailed description.

Case 1. Step-Wise PEF Inactivation of PBMCs

The ability of PEFs to selectively inactivate cells in a step-wise,size-dependent manner with increasing electric field strength andexposure time is demonstrated in this case. The stock cell suspensioncontained PBMCs suspended in IMDM at a concentration of 1.1×10⁶cells/ml. The pulsing medium was of standard physiological ionicstrength. The input cells and stock cell suspension were prepared aspreviously described. Type A test cells were used in the Case 1 trials.Pulsed electric fields, having strengths in the range 1.4-1.8 kV/cm,were applied to the specimens. The total electric field exposure timeswere in the range of 0.18-5.97 ms, and the electric field pulse lengthwas about 5.75+/−0.2 μs (FWHM). The total electric field exposure timewas varied over the noted range by varying the number of appliedelectric field pulses over a range of 30-1000 pulses. The single pulseenergy deposited to the test cell ranged from 0.54-0.88 J/pulse. Theelectric field pulses were applied at 1 Hz. The end blocks of the testcells were maintained at 35° C.+/−0.2° C. Based on Eqs. 14, 14a, and14b, the average midplane temperature varied over the range from about35.2-35.4° C. and the temperature jump per electric field pulse variedover the range 0.03-0.04° C. One stock cell control specimen and onetest cell control specimen were prepared before commencing PEFtreatments, and one test cell control specimen was prepared after allPEF treatments had been performed for the test day in question. Thecontrol and PEF treated specimens (about 5 ml each) were placed in 15 mlcentrifuge tubes after preparation, to which an approximately equalvolume of IMDM was added as previously described. These specimens werethen analyzed by flow cytometry for enumeration of viable cell types andnumbers as also previously described.

FIG. 11 presents total surviving percent of all viable cells, on they-axis, as a function of total electric field exposure time, for threedifferent electric field strengths. These results indicate that PEFlethality, expressed as surviving percent, increased with increasingelectric field strength and total electric field exposure time aspredicted by Eq. 8. Further, FIG. 11 indicates that the onset ofsignificant cell inactivation occurred at approximately 1.8 kV/cm. Thismeasured threshold strength is in reasonable agreement with thethreshold strength for the resting lymphocytes given in Table 2, whichwould be expected since the most abundant population of cells in thePBMC specimens were resting lymphocytes.

FIGS. 12a-12 d show viability scatter plots that were derived from flowcytometry assays of the specimens for this case. The results given inthese figures were generated for specimens exposed to a fixed electricfield strength of 1.8 kV/cm. FIG 12 a shows the scatter plot for thetest cell control specimen, whereas FIGS. 12b, 12 c, and 12 d show thescatter plots for 30, 100, and 300 applied electric field pulsesrespectively. The y-axes in these figures show relative propidium iodide(PI) intensity, and the x-axes show forward scattered (FSC-H) lightangles, which are proportional to cell size. High PI values areindicative of non-viable cells. High FSC-H values are large cells.Regions represented by R1 and R2 in these figures correspond to viablecells. In FIG. 12a, the viable cells in R1 and R2 form a pattern thatlooks like a pan with a handle. It can be seen that as one scans fromFIG 12 a to 12 d, the handle (i.e. viable cells in R2) disappears. Thesefigures also show that the fraction of cells that scored high for PIincreased with increasing total electric field exposure time (totalnumber of applied pulses). Thus, for a field strength of 1.8 kV/cm,FIGS. 12a-12 d indicate that the applied PEFs were selectively killingthe larger cells.

FIGS. 13a-13 c present CD11b/CD3 flow cytometry scatter plots for thecorresponding specimens shown in FIGS. 12a-12 d. FIG. 13a shows resultsfor the control specimen. Relative intensity of CD11b staining is shownon the y-axes, and relative intensity of CD3 staining is shown on thex-axes. FIGS. 13b, 13 c, and 13 d correspond to PEF treated specimensthat received 30, 100, and 300 electric field pulses of 1.8 kV/cmstrength respectively. Cells scoring high for CD11b are predominatelymonocytes. Those scoring high for CD3 are predominately T-cells. Basedon Table 2, the T-cells (a subset of lymphocytes) are typically about 7μm in size, whereas the monocytes are typically about 15 μm in size. Theupper left quadrant of FIG. 13a displays a distinct monocyte populationthat diminishes significantly as one scans from FIG. 13a to FIG. 13b toFIG. 13c and finally to FIG. 13d. In contrast, however, the T-cellpopulation (lower right quadrant) has not been reduced as significantlyas the monocyte population. Thus, the 1.8 kV/cm PEFs have selectivelydepleted the monocyte cells while preserving a significant fraction ofthe T-cells.

Case 2. PEF Enrichment of Lymphocytes over Monocytes

The ability of PEFs to enrich a PBMC specimen in lymphocytes byselective inactivation of the larger monocytes was demonstrated in thiscase. The stock cell suspension contained PBMCs suspended in IMDM at aconcentration of 1.5×10⁶ cells/ml. The pulsing medium was of standardphysiological ionic strength. The input cells and stock cell suspensionwere prepared as previously described. Type A test cells were used forthe Case 2 trials. Pulsed electric fields, having strengths in the range1.2-1.6 kV/cm, were applied to the specimens. The total electric fieldexposure times were in the range 0.15-5.70 ms, and the electric fieldpulse length was about 5.17+/−0.3 μs (FWHM). The total electric fieldexposure time was varied over the noted range by varying the number ofapplied electric field pulses over a range of 30-1000 pulses. The singlepulse energy deposited to the test cells ranged from 0.36-0.77 J/pulse.The electric field pulses were applied at 1 Hz. The end blocks of thetest cells were maintained at 35° C.+/−0.2° C. Based on Eqs. 14, 14a,and 14b, the average midplane temperature varied over the range35.1-35.3° C. and the temperature jump per electric field pulse variedover the range 0.02-0.04° C. One stock cell control specimen and onetest cell control specimen were prepared before commencing PEFtreatments, and one test cell control specimen was prepared after allPEF treatments had been performed for the test day in question. Thecontrol and PEF treated specimens (about 5 ml EACH) were placed in 15 mlcentrifuge tubes after preparation, to which an approximately equalvolume of IMDM was added as previously described. These specimens werethen analyzed by flow cytometry for enumeration of viable cell types andnumbers as also previously described.

The data in FIG. 14 is presented in the same format as shown previouslyin FIG. 11. The results indicate that PEF lethality, inverselyproportional to surviving percent, increased with increasing electricfield strength and total electric field exposure time as predicted byEq. 8. The results presented in this figure exhibit the same trends asthe data presented FIG. 11 for Case 1.

Using light scatter, viability stain gating, and CD3 and CD13 antibodyfluorescence. viable lymphocyte and monocyte cells populations wereenumerated independently. FIGS. 15a-15 j present the results in forwardscatter histogram format with relative numbers of cells on the y-axesand size on the x-axes. FIGS. 15a and 15 f show the monocyte (FIG. 15a)and lymphocyte (FIG. 15f) histograms for the control specimens.Similarly, FIGS. 15b and 15 g, 15 c and 15 h, 15 d and 15 i, and 15 eand 15 j show the monocyte and lymphocyte histograms, respectivelywithin each pair of Figs., for an electric field strength of 1.4 kV/cmand total electric field exposure times of 0.15 ms (30 pulses, FIGS. 15band 15 g), 0.33 ms (65 pulses, FIGS. 15e and 15 h), 0.70 ms (140 pulses,FIGS. 15d and 15 i), 1.50 ms (300 pulses, FIGS. 15e and 15 i). The sizedifference between the monocytes and lymphocytes can be inferred byconsidering the location of the corresponding distributions on theforward scatter axis: the monocytes are farther to the right (i.e.larger) relative to the lymphocytes (compare FIGS. 15a and 15 f forexample). Progressively comparing corresponding pairs of Figs. forrelated PEF treatment conditions and increasing electric field exposuretimes (i.e. comparing FIGS. 15a and 15 f, then FIGS. 15b and FIG. 15g,etc.), indicates that as total electric field exposure time increased,there was a large reduction in the number of monocytes, with only aminor reduction in the number of lymphocytes. (Note that the scale forthe monocyte histograms changes from FIG. 15b to FIG. 15c, and againfrom FIGS. 15d to 15 e). FIGS. 15a-15 j clearly demonstrate the sizeselective inactivation characteristics of PEFs for this cellular system.It is also noteworthy that the steepness of the tail on the right handside of the lymphocyte distributions increases with increasing electricfield exposure time an indication that the larger lymphocytes wereinactivated before the smaller ones.

The results for this case have been replotted in FIG. 16 as lymphocyteenrichment (y-axis) as a function of total electric field exposure time(x-axis) for three different electric field strengths. Lymphocyteenrichment was defined as the ratio of lymphocytes to monocytes for thecontrol specimen divided by this ratio for the PEF treated specimens.This figure clearly shows that lymphocyte enrichment increased with bothelectric field strength and total electric field exposure time. At afield strength of 1.6 kV/cm and a total electric field exposure time of˜5 ms, essentially no monocytes were detected by flow cytometry therebyyielding an essentially infinite value for lymphocyte enrichment.

Case 3. PEF Enrichment of Stem Cells in PBPC Preparations

The ability of PEFs to enrich PBPC specimens for hematopoietic stemcells by selective inactivation of the larger cells present inheterogeneous PBPC mixtures was demonstrated in this case. The stockcell suspension contained PBPCs suspended in a low ionic strengthpulsing medium (10% v/v PBS, 90% v/v isotonic sucrose solution) at aconcentration of 6.6×10⁶ cells/ml. The PBPCs were harvested frompatients, by leukopheresis, that had received G-CSF treatments aspreviously discussed. Prior to PEF treatment, the resting lymphocytes inthe PBPC preparation were activated, as previously discussed, therebystimulating these cells to their active state which nearly doubled theirsize. Type A test cells were used for the Case 3 trials. Pulsed electricfields, having strengths of 1.7, 1.8, and 1.9 kV/cm, were applied to thespecimens. The total electric field exposure time was 5.30 ms (1000applied pulses), and the electric field pulse length was 5.30 μs (FWHM).The single pulse energy deposited in the test cells ranged from0.11-0.15 J/pulse. The electric field pulses were applied at 1 Hz. Theend blocks of the test cells were maintained at about 35° C.+/−0.2° C.Based on Eqs. 14, 14a, and 14b, the average midplane temperature variedfrom about 35.0-35.1° C., and the temperature jump per electric fieldpulse varied from about 0.005-0.007° C. One stock cell control specimenand one test cell control specimen were prepared before commencing PEFtreatments, and one test cell control specimen was prepared after allPEF treatments had been performed The controls and PEF treated specimens(each about 5 ml) were placed in 15 ml centrifuge tubes afterpreparation, to which an equal volume of IMDM was added as previouslydescribed. The specimens were then analyzed by flow cytometry for cellidentification and enumeration as previously described.

Using viability (TO-PRO-3and CD34, and CD38 antibody staining, viableprimitive progenitor cells were enumerated in the control and PEFtreated specimens. Hematopoietic stem cells were identified as thoseviable cells that scored low for the viability stain, high for the CD34fluorescence marker, and low for the CD38 marker (i.e., CD34+/CD38− orCD34 single positive cells). The results for this case are presented inFIG. 17 in bar chart format. The shaded bars in FIG. 17 correspond tothe surviving percent of all cells in the suspension. The unshaded barscorrespond to stem cell enrichment, which was defined as the ratio ofviable stem cells to total viable cells in the PEF treated specimensnormalized by the same ratio for the control specimens. FIG. 17 showsthat the total surviving percent of cells decreased with increasingelectric field strength. However, stem cell enrichment increased withincreasing electric field strength. In fact, at 1.9 kV/cm, enrichmentapproached 1 log. Significantly, the enrichment increased inverselyproportional to the decrease in total surviving percent. This indicatesthat the stem cell population was being preserved and demonstrates theability of PEF's to enrich PBPC specimens for stem cells.

It should be re-emphasized that these results were obtained underconditions that were not optimized for stem cell enrichment. Morespecifically, the electric field waveform shape used for this case (seeFIG. 10) was Gaussian in shape, rather than rectangular, and the lengthof the pulse (5 μs) was very short. As discussed previously, theelectric field waveform shape is preferably rectangular (with very shortrise/fall times and with an essentially constant field strength betweenrise and fall) for obtaining more optimal size selectivity. Also, asdiscussed previously, short electric field pulses, such as the 5 μspulses used in the present case are generally less effective for cellinactivation than pulse durations greater than about 10 μs.

Case 4. PEF Purging of CMK Tumor Cells in PBMCs

This case demonstrates the ability of PEFs to purge PBMC suspensions oftumor cells. The stock cell suspension included PBMCs and CMK tumorcells suspended in a low ionic strength pulsing medium (10% v/v PBS, 90%v/v isotonic sucrose solution) at 2.5×10⁶ cells/ml. The CMK tumor cellsrepresented about 14% of the total number of cells. The CMKs are amegakaryocyte line whose size approximates epithelial tumor cell types.The PBMCs and CMKs were prepared as previously described. Type A testcells were used for the Case 4 trials. Pulsed electric fields, havingstrengths in the range 1.2-1.8 kV/cm, were applied to the specimens. Thetotal electric field exposure time was 3.6 ms (1000 applied pulses), andthe electric field pulse length was 3.6 μs (FWHM). The single pulseenergy deposited to the test cells ranged from 0.04-0.10 J/pulse, andthe electric field pulses were applied at 1 Hz. The end blocks of thetest cells were maintained at 35° C.+/−0.2° C. Based on Eqs. 14, 14a,and 14b, the average midplane temperature varied from about 35.02-35.04°C. and the temperature jump per electric field pulse varied from about0.002-0.005° C. One stock cell control specimen and one test controlspecimen were prepared before commencing PEF treatments, and one testcell control specimen was prepared after all PEF treatments had beenperformed. The controls and PEF treated specimens (each about 5 ml) wereplaced in 15 ml centrifuge tubes after preparation, to which anapproximately equal volume of IMDM was added as previously described.The specimens were then analyzed by flow cytometry for cellidentification and enumeration as previously described. FIGS. 18a-18 fshow the flow cytometry data from the analysis of the input cells forthis case. FIGS. 18a, 18 c. and 18 e are the light scatter plots (noviability stain gating) for the CMK cells alone, the PBMCs alone, andthe CMK/PBMC mixture, respectively. FIGS. 18b, 18 d, and 18 f areviability stain histograms for the CMKs alone, the PBMCs alone, and theCMK/PBMC mixture respectively. Cells staining for the viability stain(TO-PRO-3with an intensity greater than 10² were considered dead. FIG.18b indicates that the CMK cells had a component (i.e. that populationabove 10² on the x-axis) that was either dead or represented celldebris. These dead cells or debris showed up in the light scatter plot(FIG. 18a) as a band of dots that extend up and to the right from theorigin. The viable CMK population appeared as a cluster of dots that iscentered vertically in FIG. 18a and slightly to the right of centerhorizontally. FIG. 18c indicates that were three clusters of dots forthe PBMCs. The cluster near the origin represents fine cell debris. Thenext cluster to the right represents the lymphocyte population, and thecluster furthest to the right represents the monocyte population. Thelymphocyte, monocyte, and CMK clusters are evident in FIG. 18e.

FIGS. 19a-19 f qualitatively illustrate the effect of applying 1000electric field pulses, each with a strength of 1.8 kV/cm, to theCMK/PBMC mixture. FIGS. 19a, 19 b, and 19 c are a light scatter plot,CD14/CD45 bivariate plot, and TO-PRO-3 viability histogram, respectivelyfor the control specimen. FIGS. 19d, 19 e, and 19 f present the samecorresponding information as FIGS. 19a, 19 b, and 19 c respectively, forthe specimen treated with 1000 electric field pulses, each having astrength of 1.8 kV/cm. As in FIG. 18e, the lymphocyte, monocyte, and CMKpopulations are evident in FIG. 19a. Circled region R1 (in FIGS. 19a and19 d) enclose the CMK population. Bracketed region R3 (in FIG. 19c and19 f) defines viable cells as judged by TO-PRO-3 viability stainingintensity. Scatter plots shown in FIGS. 19b (control) and 19 e (PEFtreated specimen) were gated on both R1 and R3, so the cells appearingin these two figures represent viable cells in the R1 light scattercompartment, which is dominated by the CMK tumor cells. The lower rightquadrant of FIG. 19b shows a well defined CMK population. The upperright quadrant, however, shows that the R1 region also contained a smallnumber of monocytes. FIG. 19e shows that both the CMKs and monocyteswere eliminated by application of 1000 electric field pulses of 1.8kV/cm strength. However, the light scatter plot (FIG. 19d) for the PEFtreated specimen indicates that the specimen still contained healthylymphocyte and monocyte populations. The change in the TO-PRO-3viability histograms (FIG. 19c and FIG. 19f) also indicates that onlythe CMK population had been affected by PEF treatment (the central peakin FIG. 19c, which is missing in FIG. 19f corresponds to the CMKpopulation, which was derived by considering FIG. 18b).

FIGS. 20a-20 f examine the effect of PEFs on the lymphocyte population.FIGS. 20a, 20 b, and 20 c are light scatter, CD3/CD19 bivariate plots,and TO-PRO-3 viability histograms, respectively, for the controlspecimen. FIGS. 20d, 20 e, and 20 f are the light scatter, CD3/CD19bivariate plots, and TO-PRO-3 viability histograms, respectively, forthe specimen that was treated with 1000 electric field pulses, eachhaving a strength of 1.8 kV/cm. In FIG. 20a and FIG. 20d, regionslabeled R4, R6, and R7 correspond to the light scatter compartments thatenclose the lymphocytes, monocytes, and CMKs, respectively. The TO-PRO-3viability histograms in FIG. 20c and FIG. 20f were gated on R4, whichmeans that only events in the lymphocyte compartments in the lightscatter plots are displayed in FIG. 20c and FIG. 20f. FIGS. 20b and 20 eare gated on R8 (the TO-PRO-3 viability ranges in either FIG. 20c orFIG. 20d) and R4 (the lymphocyte compartment in either FIG. 20a and FIG.20d). Thus, FIG. 20b and FIG. 20e display only the viable cells from thelymphocyte light scatter compartments. The conjugate monoclonal antibodyfluorescence marker CD3 stains T-cells, a subset of lymphocytes. Thefluorescence marker CD19 stains B-cells. Thus, the upper left quadrantsin FIG. 20b and FIG. 20e contain viable T-cells, whereas the lower rightquadrants contain viable B-cells. Comparison of FIG. 20b and FIG. 20eindicates there was little difference in the abundance of viable T- andB-cells for the control and PEF treated specimens. However, FIGS. 19band 19 e clearly show that the CMK cells had been almost entirelyeliminated under the same PEF conditions.

FIG. 21 presents the full set of data collected for this case. In thisfigure, the surviving percents of the relevant cell types (y-axis) arepresented as a function of electric field strength (x-axis). Recall thatthe total electric field exposure time was constant for each fieldstrength and was produced by applying 1000 electric field pulses. Thetotal electric field exposure time was 3.6 ms. This figure clearly showsthat an almost 2-log reduction in CMKs was achieved at 1.8 kV/cm withoutimpacting the viability of the lymphocytes. Also, it is evident that themonocytes were just beginning to be affected at 1.8 kV/cm, since themonocyte surviving percent curve is beginning to decrease at this fieldstrength. Comparing the PBMC total surviving percent curves presented inFIGS. 11 and 14 with the lymphocyte surviving percent curve in FIG. 21,shows the lethal effects of PEFs occurs at lower electric fieldstrengths in FIGS. 11 and 14. As already indicated, the resultspresented in these examples have been acquired under non-optimizedconditions. The differences in PEF efficacy found by comparing FIGS. 11and 14 with FIG. 21 may be due to pulse length and pulsing medium ionicstrength differences. The pulse length for the results in FIG. 21 wasapproximately 30% shorter than for the results in FIGS. 11 and 14.Further, the results in FIG. 21 were obtained using a low ionic strengthpulsing medium, rather than a standard physiological ionic strengthmedium as used for the results presented in FIGS. 11 and 14.

As noted, the pulsing medium used for this case was a low ionic strengthpulsing buffer (10% v/v PBS, 90% v/v isotonic sucrose solution). Thislow ionic strength pulsing medium was used for two reasons. First, aspreviously discussed, application of PEFs to cells in a low ionicstrength pulsing medium, followed by resuspension in a standardphysiological strength buffer, can lead to more extensive fragmentationof the of the PEF porated cells. It was observed during PEF cellselection experiments that post-PEF specimens that were treated withPEFs in a low ionic strength pulsing medium, followed by resuspension ina higher ionic strength medium, included far fewer trypan blue stainedcells than when treated with PEFs under conditions where the pulsingbuffer was of standard physiological ionic strength, even though thereductions in viable cells were comparable. This result indicates thatthe combination of a low ionic strength pulsing buffer and a higherionic strength post-PEF resuspension buffer led to greater fragmentationof the PEF porated cells by colloidal osmotic cell lysis than forconditions where the pulsing buffer was of standard physiological ionicstrength. Secondly, a lower ionic strength pulsing medium requires lowerenergy input to achieve the same electric field strengths.

Case 5. PEF Inactivation Characteristics of Breast Tumor Cells

The efficacy of PEF inactivation of breast tumor cells was investigatedin this case. The stock cell suspension contained only breast tumorcells (MCF-7), which were suspended in a low ionic strength pulsingmedium (10% v/v PB5, 90% v/v isotonic sucrose solution) at a totalconcentration of 1.2×10⁶ cells/ml. The MCF-7s were prepared aspreviously described. Type B test cells were used for the Case 5experiments. Pulsed electric fields, having field strengths in the rangeof 1.0-2.0 kV/cm were applied to the specimens. Two electric field pulselengths were used for this case: 3.50 and 5.25 μs (FWHM). The totalelectric field exposure time was 3.5 ms (1000 applied pulses) for theshortest electric field pulse length, whereas the total electric fieldexposure time for the longest electric field pulse was 4.7 ms (900pulses). The slight reduction in pulse number for the longer pulselength experiments was included to keep the total electric fieldexposure time for the longer pulse length experiments, based on thepulse length at 95% of the peak electric field strength, approximatelythe same as for the shorter pulse length experiments. The single pulseenergy deposited to the test cells was in the range of 0.003-0.020J/pulse. The electric field pulses were applied at 1 Hz. The end blocksof the test cells were maintained at about 35° C.+/−0.2° C. Based onEqs. 14, 14a, and 14b, the average midplane temperature varied over therange from 35.01-35.04° C., and the temperature jump per electric fieldpulse varied over the range 0.001-0.007° C. One stock cell controlspecimen and test cell control specimen were prepared before commencingPEF treatments, and one test cell control specimen was prepared afterall PEF treatments had been performed for each set of tests. Thecontrols and PEF treated specimens (each about 0.72 ml) were placed in15 ml centrifuge tubes, to which about 5 ml of IMDM was added aspreviously described. These specimens were then analyzed by flowcytometry for enumeration of viable MCF-7s, also as previouslydescribed.

For this case, viable MCF-7s were identified as those cells thatfluoresced dimly for both propidium iodide (PI) and Annexin-V stains.FIG. 22 presents results typical of PEF inactivation experiments usingthe MCF-7 cell line. More specifically, this figure illustrates thecombined effect of increasing electric field pulse length and exposuretime, as well as electric field strength. The curve shown by the dashedline represents data obtained by applying 1000 electric field pulses,each pulse having a 3.5 μs FWHM pulse length. The curve shown by thesolid line represents data obtained by applying 900 electric fieldpulses, each pulse having a 5.3 μs FWHM pulse length. Thus, the totalelectric field exposure time for the dashed curve was 3.5 ms, whereasthe total electric field exposure time for the solid curve was 4.7 ms.

FIG. 22 clearly shows that increasing PEF pulse length and exposure timeresulted in increased tumor cell purging efficacy. Further, the 5.3 μspulse length produced about a 2.3 log reduction in viable tumor cells.It is important to note that the 2.3 log reduction shown maysignificantly understate the efficacy of the PEF process. Opticalmicroscopy indicated that the input cell population included clumps ofagglomerated cells, each clump containing from about 3-10 cells. Thus,the number of viable MCF-7 cells in the control specimens, which wereused to normalize the data for viable cells contained in the PEF-treatedspecimens, could be low by about a factor of 3-10. Thus, the survivingpercents reported for the PEF-treated specimens could be high by afactor of 3-10. In addition, application of PEFs to this cell line hadtwo effects: 1) the PEFs breakup the clumps of aggregated cells, and 2)inactivation of the cells. The breakup of cells was clearly observed bythe increase in total events recorded by the cytometer. Total eventswere observed to increase by at least a factor of two once PEFs had beenapplied to a specimen. It is also believed the inflection points in thecurves of FIG. 22 could be due to an increase in cell numbers due todisaggregation by the PEFs, followed by subsequent inactivation.Significantly, some of the cells in clumps will be shielded from thePEFs until such clumps are completely reduced to monodispersed cells.Once monodispersed, they can then be inactivated by the PEFs. Thus thepresence of clumped cells in the cell suspension before treatment wouldimply that not all of the cells in the specimens experienced the sameeffective electric field exposure time. Thus, increasing the electricfield pulse length, may be an effective way to lead to more efficientPEF clump disaggregation, which, in turn, could allow the use of lowerfield strengths and possibly shorter exposure times to achieveinactivation of cells that tend to clump, like the MCF-7 tumor cells.The MCF-7 tumor cell inactivation results presented here show that atleast a 2.3 log reduction in this breast cancer line was achieved at PEFconditions which led to a 1 log enrichment of stem cells, withoutsignificant loss of viable stem cells, in Case 3 presented above.

Case 6. Enzymatic Removal of a Glycocalyx Membrane Layer

A small fraction of the cells within a given epithelial cell line, suchas the MCF-7 line, secrete a mucopolysaccharide (mucin), which can coatthe cell plasma membrane. This mucin coat can function to increase theeffective thickness of the membrane of these cells, which can, in turn,require higher electric field strengths for their inactivation. Theinventors have experimentally determined, using standard mucin stainsand optical microscopy, that a fraction of the MCF-7 cells have a mucincoat and that the coat can be removed by enzyme digestion. To achievethis, the MCF-7s were subjected to a hyaluronidase digestion protocoljust prior to their suspension in the pulsing medium. The mucindigestion protocol involved resuspending the trypsinized cells in adigestion solution (500 ug/ml hyaluronidase, Sigma, H4272 30 mg; 94 mMpotassium phosphate monobasic, Sigma, P8416; 6 mM Sodium phosphatedibasic, Sigma, S-5136) and incubating the solution for 30 minutes at37° C. Using standard techniques for mucin staining under opticalmicroscopy it was found that this digestion protocol essentiallyentirely removed the extracellular mucin coats.

While the invention has been shown and described above with reference tovarious embodiments and specific examples, it is to be understood thatthe invention is not limited to the embodiments or examples describedand that the teachings of this invention may be practiced by one skilledin the art in various additional ways and for various additionalpurposes.

What is claimed is:
 1. A suspension of cells obtained by practicing amethod comprising the steps of: a. providing a biological samplecomprising a cell suspension having a given cell population of at leasta first and a second cell type, at least said first cell type beingnucleated, wherein said first and second cell types have a difference inat least one property affecting a characteristic electroporationthreshold; b. increasing the proportion of viable cells in the cellsuspension that are viable cells of said second cell type withoutexposing the cell population to exogenous toxins by: i. subjecting thesample to electric field conditions sufficient to porate a substantialfraction of cells of said first cell type while maintainingsubstantially viable cells of said second cell type; and ii. selectivelyinactivating at least 90% of the porated cells of said first cell typein the sample subjected to the electric field conditions in step (b)(i),while maintaining substantially viable cells of said second cell type inthe sample subjected to the electric field conditions in step (b)(i). 2.The cell suspension as in claim 1, wherein in step b the proportion ofviable cells in the cell suspension that are viable cells of said secondcell type is increased without exposing the cell population to exogenousantibodies to cell surface markers.
 3. The cell suspension as in claim1, wherein steps b(i) and b(ii) are performed simultaneously.
 4. Thesuspension as in claim 3, wherein said at least one property affecting acharacteristic electroporation threshold comprises cell size.
 5. Thesuspension as in claim 4, wherein the electric field conditions aresufficient to inactivate cells of the sample having a characteristicsize greater than a selected threshold size by causing irreversibleporation of the cell membrane.
 6. The suspension as in claim 5, whereina substantial fraction of the cells inactivated by said electric fieldconditions undergo irreparable cell lysis during the inactivating step.7. The cell suspension as in claim 1, wherein both said first and secondcell types are nucleated.
 8. The suspension as in claim 1, wherein saidat least one property affecting a characteristic electroporationthreshold comprises cell size.
 9. The suspension as in claim 1, whereinat least 99% of the porated cells of said first cell type areselectively inactivated in step (b)(ii).
 10. The suspension as in claim9, wherein at least 99.99999% of the porated cells of said first celltype are selectively inactivated in step (b)(ii).
 11. The suspension asin claim 10, wherein essentially all of the porated cells of said firstcell type are selectively inactivated in step (b)(ii).
 12. A cellsuspension comprising: a plurality of biological cells suspended in aliquid including a first population of cells having a maximumcharacteristic size not more than a predetermined value that aresubstantially viable and a second population of nucleated cells having amaximum characteristic size greater than said predetermined value thatare substantially non-viable, said cell suspension being essentiallyfree of exogenous toxins and being obtained from a precursor cellsuspension comprising substantially viable cells, which precursor cellsuspension contains as subpopulations said first and second populationsof cells, said cell suspension being obtained by increasing theproportion of viable cells in said precursor cell suspension that areviable cells of said first population of cells by: subjecting theprecursor cell suspension to an electric field of a magnitude andduration sufficient to porate a substantial fraction of cells of saidsecond cell population in the precursor suspension and inactivate atleast 90% of cells of said second population porated by the electricfield, while maintaining substantially viable cells of said firstpopulation.
 13. The cell suspension as in claim 12, wherein saidbiological cells are derived from an animal.
 14. The cell suspension asin claim 13, wherein said animal is a human.
 15. The cell suspension asin claim 12, wherein said second population of cells include cancercells.
 16. The cell suspension as in claim 12, wherein said firstpopulation of cells include stem cells.
 17. The cell suspension as inclaim 16, wherein said stem cells include pluripotent stem cells. 18.The cell suspension as in claim 16, wherein said stem cells comprise atleast one of the following: mesenchymal stem cells; embryonic stemcells; epithelial stem cells; gut stem cells; liver progenitor cells;endocrine progenitor cells; skin stem cells; or neural stem cells. 19.The cell suspension as in claim 16, wherein said stem cells includehematopoietic stem cells.
 20. The cell suspension as in claim 19,further comprising at least one population of cells selected from thegroup consisting of: colony forming cells for granulocytes andmacrophages (CFC-GM), colony forming cells for erythrocytes (BFU-E),colony forming cells for eosinophils (CFC-Eo), multipotent colonyforming cells (CFC-GEMM), and immature lymphoid precursor cells.
 21. Thecell suspension as in claim 16, wherein said stem cells include stemcells that are essentially free of cell surface CD34 markers.
 22. Thecell suspension as in claim 12, wherein the cell suspension isessentially free of exogenous antibodies to cell surface markers. 23.The cell suspension as in claim 12, wherein said first population ofcells is nucleated.
 24. The suspension as in claim 12, wherein asubstantial fraction of the cells inactivated by said electric fieldundergo irreparable cell lysis during the subjecting step.
 25. Thesuspension as in claim 12, wherein at least 99% of the cells of saidsecond population are inactivated in the subjecting step.
 26. Thesuspension as in claim 25, wherein at least 99.99999% of the cells ofsaid second population are inactivated in the subjecting step.
 27. Thesuspension as in claim 26, wherein essentially all of the cells of saidsecond population are inactivated in the subjecting step.